Synthesis and DNA/RNA Binding Properties of Conformationally Constrained Pyrrolidinyl PNA with a Tetrahydrofuran Backbone Deriving from Deoxyribose

Article abstract of DOI:10.1021/acs.joc.5b00890

Sugar-derived cyclic β-amino acids are important building blocks for designing of foldamers and other biomimetic structures. We report herein the first synthesis of a C-activated N-Fmoc-protected trans-(2S,3S)-3-aminotetrahydrofuran-2-carboxylic acid as a building block for Fmoc solid phase peptide synthesis. Starting from 2-deoxy-d-ribose, the product is obtained in a 6.7% overall yield following an 11-step reaction sequence. The tetrahydrofuran amino acid is used as a building block for a new peptide nucleic acid (PNA), which exhibits excellent DNA binding affinity with high specificity. It also shows preference for binding to DNA over RNA and specifically in the antiparallel orientation. In addition, the presence of the hydrophilic tetrahydrofuran ring in the PNA structure reduces nonspecific interactions and self-aggregation, which is a common problem in PNA due to its hydrophobic nature.

Full text of DOI:10.1021/acs.joc.5b00890

Article
pubs.acs.org/joc
Synthesis and DNA/RNA Binding Properties of Conformationally
Constrained Pyrrolidinyl PNA with a Tetrahydrofuran Backbone
Deriving from Deoxyribose
Pitchanun Sriwarom, Panuwat Padungros, and Tirayut Vilaivan*
Organic Synthesis Research Unit, Department of Chemistry, Faculty of Science, Chulalongkorn University, Phayathai Road,
Patumwan, Bangkok 10330, Thailand
S
* Supporting Information
ABSTRACT: Sugar-derived cyclic β-amino acids are impor-
tant building blocks for designing of foldamers and other
biomimetic structures. We report herein the first synthesis of a
C-activated N-Fmoc-protected trans-(2S,3S)-3-aminotetrahy-
drofuran-2-carboxylic acid as a building block for Fmoc solid
phase peptide synthesis. Starting from 2-deoxy-D-ribose, the
product is obtained in a 6.7% overall yield following an 11-step
reaction sequence. The tetrahydrofuran amino acid is used as a building block for a new peptide nucleic acid (PNA), which
exhibits excellent DNA binding affinity with high specificity. It also shows preference for binding to DNA over RNA and
specifically in the antiparallel orientation. In addition, the presence of the hydrophilic tetrahydrofuran ring in the PNA structure
reduces nonspecific interactions and self-aggregation, which is a common problem in PNA due to its hydrophobic nature.
Scheme 1. Structures of acpcPNA and atfcPNA
INTRODUCTION
■
Foldamers are synthetic oligomeric molecules that can mimic
naturally occurring biological macromolecules by folding into
well-defined secondary structures such as helices, turns, or
sheets in solution states.1 Amino acids, especially cyclic β-amino
acids,2 constitute an important class of building blocks for
foldamers.3 A diverse range of secondary structures were
obtained from simple cyclic β-amino acids building blocks with
different ring size, stereochemistry, and substituents.4 The
ability to precisely program the folding pattern is not only
important from the molecular design aspect but also leads to
desirable biological functions. Carbohydrate-derived amino
acids5 have recently attracted much interest as potential building
blocks for novel foldamers.6 It can be readily obtained from
inexpensive sources in stereochemically defined configurations
and possess a number of functional groups that can be fine-
tuned as desired.
Deoxyribonucleoside-derived cyclic β-amino acids have been
used in designing nucleobase-containing foldamers,7 some of
which showed promising DNA and RNA binding properties.7d
These foldamers can therefore be regarded as a new class of
peptide nucleic acid (PNA).8,9 Our research group had
introduced a series of conformationally constrained pyrrolidinyl
peptide nucleic acids derived from alternating nucleobase-
modified D-proline and cyclic β-amino acids.10,11 A representa-
tive member of such pyrrolidinyl PNA is acpcPNA (Scheme 1)
which carries trans-(1S,2S)-2-aminocyclopentanecarboxylic acid
(ACPC) in the backbone.10b,d This acpcPNA showed some
distinct properties from DNA and commercial PNA that could
be useful for many applications.12 A recent study on ring
homologues suggested that four- or five-membered ring cyclic β-
amino acids with specific configurations are essential to provide
a balance between rigidity and flexibility that allows the PNA to
adopt a DNA-binding conformation.10e In addition to ACPC,
other five-membered ring heterocyclic β-amino acids had been
successfully incorporated into pyrrolidinyl PNA, including 1-
aminopyrrolidine-2-carboxylic acid10a and 3-aminopyrrolidine-
4-carboxylic acid.10c
In view of the structural similarity between 3-amino-
tetrahydrofuran-2-carboxylic acid (ATFC) and ACPC, it is not
surprising that oligomers of ATFC and its ring-substituted
derivatives can also form well-defined helical foldamers.13
Oligomers of trans-ATFC formed a 12-helix similar to trans-
ACPC. On the other hand, the cis-isomer adopted a 14-helix
structure instead of the extended sheetlike structure of cis-
ACPC, which was explained based on molecular modeling
studies by a more favorable interaction between the less
sterically hindered ring of ATFC and the backbone amide
group.13a We propose that replacement of ACPC in the
acpcPNA backbone with trans-ATFC should provide a new
pyrrolidinyl PNA that can still retain the excellent nucleic acid
Received: April 21, 2015
Published: June 17, 2015
© 2015 American Chemical Society
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Scheme 2. Structural Assignment of Methyl Glycoside Mixture by Conversion into Their 1-Deoxy Perbenzoylated Derivatives
binding characteristics of acpcPNA. Moreover, it is expected
that the more hydrophilic nature of tetrahydrofuran ring in
ATFC as opposed to the cyclopentane ring in ACPC should be
beneficial in reducing self-aggregation and nonspecific inter-
actions occasionally observed in acpcPNA and other PNA
systems due to their uncharged and hydrophobic peptide
backbone.14 Herein, we report the synthesis of a new
pyrrolidinyl PNA with an ATFC backbone (will be named
atfcPNA, Scheme 1) and a comparison with acpcPNA in terms
of DNA/RNA binding properties and nonspecific binding
behavior.
Scheme 3. Synthesis of SS-ATFC Starting from 2-Deoxy-D-
ribose (2)
RESULTS AND DISCUSSION
■
Synthesis of Deoxyribose-Derived Cyclic β-Amino
Acid (SS-ATFC). Our previous reports on the effects of
stereochemistry of acpcPNA on DNA binding11 suggests that
the appropriately protected (2S,3S) enantiomer of the trans-
ATFC (SS-ATFC) is required as a key building block.
Interestingly, this particular configuration of ATFC is unknown
in the literature despite some reports on the remaining
stereoisomers.13a We proposed that the required SS-ATFC
monomer 1 should be readily synthesized starting from
commercially available 2-deoxy-D-ribose (2). The key steps
involved reductive removal of the anomeric hydroxyl group,
stereospecific introduction of the amino group by a double
inversion of configuration at C-3, and oxidation of the C-5
alcohol to the carboxylic acid.
With this synthetic strategy in mind, the anomeric hydroxyl
group of 2-deoxy-D-ribose (2) was first converted to its
methylglycoside derivative in the presence of an acid catalyst
(Scheme 2).15,16 It should be noted that the reaction time
played an important role for the outcome of the reaction.17 1H
NMR analysis of the product mixture was simplified by
perbenzoylation followed by reduction of the anomeric methyl
ether with BF3·Et2O/Et3SiH.18 Prolonged treatment of 2 with
catalytic methanesulfonic acid in MeOH (12 h) provided a 1.5:1
mixture of furanoside 3 and the thermodynamically more stable
pyranoside. However, immediate quenching of the reaction after
consumption of the starting material (ca. 30 min) afforded the
methylfuranoside 3 as the major product (>80% of the crude
reaction mixture).
the furanoside 4 was tosylated to afford 5 in 83% yield, and the
anomeric methoxy group was successfully reduced to provide
intermediate 6 in 86% yield.
We next focused on the nucleophilic displacement of the
tosylated furanoside 6. The nitrite ion (NaNO2) is relatively
overlooked as an oxygen nucleophile for the stereochemical
inversion of a hydroxyl group. It has been successfully employed
in the inversion of various alcohols including carbohydrate
derivatives.20 The nucleophilic substitution of furanoside 6 with
excess NaNO2 at 120 °C for 5 h provided a 1:1 mixture of the
expected inverted alcohol 7 and a side-product 7′ resulting from
migration of the p-toluoyl group to the neighboring inverted C-3
hydroxyl group in moderate yield. Shortening the reaction time
to 2 h gave a 4.5:1 regioisomeric mixture of 7:7′ in 58% yield.
The sterically hindered C-3 hydroxyl group of 7 is much less
reactive than the free C-5 hydroxyl group of 7′, and therefore,
benzoylation of the mixture facilitated the chromatographic
purification of unreacted 7 from benzoylated 7′ in 89% recovery
yield.
With the optimized conditions in hand, 2-deoxy-D-ribose (2)
was smoothly converted to methylfuranoside 3 followed by
selective protection19 of the C-5 primary hydroxyl group as the
p-toluoyl ester to afford furanoside 4 in 67% yield over 2 steps
(Scheme 3). Removal of the anomeric methoxy group by
treatment with BF3·Et2O/Et3SiH was previously reported on a
fully protected 2-deoxyribose derivative,18 and thus it was
uncertain if these conditions would be compatible with the
unprotected C-3 hydroxyl group of furanoside 4. It was
envisioned that the C-3 hydroxyl group could be “protected”
as a sulfonate ester prior to the anomeric reduction. Gratifyingly,
The C-3 hydroxyl group of 7 was next converted to the
mesylate 8 in 90% yield. It is worth noting that attempts to
perform Mitsunobu reaction of furanoside 4 with MeOTs or
methanesulfonic acid to yield the mesylate with inverted
configuration at C-3 gave only complex mixtures.21 Reaction
of 8 with excess NaN3 at 90−100 °C for 2 h gave the expected
azide 9 in 95% yield. Hydrogenation of 9 in the presence of
Boc2O gave the Boc-protected amine 10 in 71% yield as a white
solid. Hydrolysis of p-toluoyl group in 10 followed by BAIB-
TEMPO22 oxidation gave the Boc-protected amino acid 12 in
high yield. Treatment with trifluoroacetic acid (TFA) liberated
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Table 1. Sequence and Characterization Data of atfcPNA
a
b
c
d
PNA
T9
sequence (N→C)
N/C-terminal modification
tR min
30.6
25.1
m/z (calcd)
m/z (found)
yield %
TTTTTTTTT
Ac/LysNH2
Ac/LysNH2
Flu/LysNH2
Bz/LysNH2
Bz/LysNH2
3198.2
3578.6
3894.8
3630.6
3617.6
3197.1
3578.4
3893.3
3630.7
3617.9
44
22
15
20
10
M10
GTAGATCACT
GTAGATCACT
TATGTACTAT
GCTACGTCGC
M10Flu
M10AT
M10CG
28.4, 29.2
29.1
28.1
a
HPLC conditions: C18 column, 4.6 × 50 mm, 3 μ, gradient 0.1% TFA in H2O:MeOH 90:10 for 5 min then linear gradient to 10:90 over 30 min,
b
c
d
flow rate 0.5 mL/min, 260 nm. Average mass of M + H+. MALDI-TOF. Isolated yield after HPLC purification, spectrophotometrically
determined.
Table 2. Tm of atfcPNA/acpcPNA and DNA/RNA
a
a,b
PNA (N→C)
DNA or RNA (5′→3′)
Tm atfcPNA (ΔTm)
Tm acpcPNA (ΔTm)
note
GTAGATCACT (M10)
dAGTGATCTAC
dAGTGCTCTAC
dAGTGGTCTAC
dAGTGTTCTAC
dCATCTAGTGA
rAGUGAUCUAC
rAGUGCUCUAC
rAGUGGUCUAC
rAGUGUUCUAC
rCAUCUAGUGA
dATAGTACATA
dATAGAACATA
dATAGCACATA
dATAGGACATA
dATACATGATA
rAUAGUACAUA
rAUACUAGAUA
dGCGACGTAGC
dGCGAAGTAGC
dGCGAGGTAGC
dGCGATGTAGC
dCGATGCAGCG
rGCGACGUAGC
rCGAUGCAGCG
52.5
53.3
complementary DNA
mismatched DNA
mismatched DNA
mismatched DNA
parallel DNA
23.4 (−29.1)
23.4 (−29.1)
25.4 (−27.1)
<20
23.8 (−29.5)
23.9 (−29.4)
29.4 (−23.9)
<20
36.0
42.3
complementary RNA
mismatched RNA
mismatched RNA
mismatched RNA
parallel RNA
<20
23.6 (−18.7)
24.8 (−17.5)
<20
<20
<20
<20
<20
TATGTACTAT (M10AT)
49.6
54.2
complementary DNA
mismatched DNA
mismatched DNA
mismatched DNA
parallel DNA
25.4 (−24.2)
30.2 (−19.4)
<20
23.4 (−30.8)
30.2 (−24.0)
24.4 (−29.8)
<20
<20
29.2
32.6
complementary RNA
parallel RNA
<20
<20
GCTACGTCGC (M10CG)
56.4
54.5
complementary DNA
mismatched DNA
mismatched DNA
mismatched DNA
parallel DNA
23.4 (−33.0)
31.2 (−25.2)
36.0 (−20.4)
<20
<20
30.2 (−24.3)
35.1 (−19.4)
<20
41.8
48.0
complementary RNA
parallel RNA
24.4 (−17.4)
39.2 (−8.8)
a
All Tm were measured at PNA = 1 μM, 100 mM NaCl, 10 mM sodium phosphate buffer, pH 7.0, heating rate 1 °C/min; and ΔTm = Tm−
b
complementary hybrid
Tm
The Tm data of acpcPNA are provided for comparison purposes. Except for the mismatched hybrids of M10AT and M10CG, Tm
data of all acpcPNA hybrids were taken from ref 10d. Mismatch positions in DNA sequence are indicated by the underline.
the free amino acid which was further reacted with FmocOSu to
give the desired Fmoc-protected amino acid 13 in 87% yield
over 2 steps. Finally, the Fmoc-protected amino acid 13 was
activated as its pentafluorophenyl (Pfp) ester 1 in 65% yield.
The presence of the Pfp group was confirmed by 19F NMR
analysis. In summary, the target cyclic β-amino acid spacer (SS-
ATFC) 1 was successfully synthesized from commercially
available 2-deoxy-D-ribose (2) in a straightforward fashion over
11 steps in 6.7% overall yield.
Synthesis of atfcPNA. Four different sequences of atfcPNA
(T9, M10, M10AT, and M10CG) were synthesized from the
activated SS-ATFC spacer 1 and the four Fmoc-protected
pyrrolidinyl PNA monomers (ABz, T, CBz, and GIbu) following
the stepwise coupling protocol for Fmoc solid phase synthesis of
PNA previously developed in our laboratory (Table 1).10b The
atfcPNA sequences were end-capped at the N-termini by
acetylation or benzoylation. In addition, an atfcPNA sequence
(M10) was also end-capped with 5(6)-carboxyfluorescein to
yield a M10Flu sequence in order to study nonspecific
interactions. All atfcPNAs were purified by reverse phase
HPLC and their identities were confirmed by MALDI-TOF
mass spectrometry. In all cases, the PNAs were obtained with
>90% purity, except for the M10Flu sequence which is split into
two peaks on the HPLC chromatogram due to the presence of
two carboxyfluorescein isomers. Isolated yields in the range of
10−44% were obtained, depending on the sequence. The poor
isolated yields, which are typical for most solid phase syntheses,
are attributed to the loss of product during HPLC purification
due to separation issues rather than the synthetic efficiency. All
PNAs are readily soluble in water (>1 mM). The sequences and
characterization data of all atfcPNA are summarized in Table 1.
DNA and RNA Binding Properties of atfcPNA.
Preliminary DNA binding studies were performed on the T9
sequence. Since homopyrimidine/homopurine PNA sequences
are known to form triplex structures, it is important to first
determine the stoichiometry of binding. This was accomplished
by UV titration (Figure S22 of the Supporting Information),
which clearly confirms a 1:1 stoichiometry of atfcPNA:DNA as
shown by the inflection point at ca. 50 mol % DNA. This is in
agreement with other pyrrolidinyl PNA, whereby no (PNA)2·
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DNA triplex formation could be observed, presumably due to
the bulkiness of the pyrrolidinyl PNA backbone.
Melting temperature measurements by UV spectrophotom-
etry suggest that the T9 atfcPNA binds cooperatively to its
complementary DNA (dA9), giving well-defined melting curves
with melting temperatures (Tm) of 60.3 and 64.1 °C (in the
presence and absence of 100 mM NaCl, respectively). These Tm
figures are somewhat lower than the corresponding T9
acpcPNA (Tm = 72.5 and >76.8 °C, in the presence and
absence of 100 mM NaCl, respectively).10d,e Nevertheless, it is
clear that T9 atfcPNA can form a stable hybrid with its
complementary DNA. In the presence of a mismatched base in
the DNA strand, the Tm was dramatically decreased (−21.6,
−21.9, and −31.8 °C for pT-dC, pT-dT, and pT-dG
mismatches, respectively). The large decrease of Tm suggests
that the atfcPNA binds to DNA with high specificity similar to
that of acpcPNA.
Figure 1. CD spectra of (a) T9 atfcPNA (blue), dA9 (black), and their
1:1 hybrid (green) and (b) M10CG atfcPNA (blue) and its hybrids
with antiparallel DNA (green) or antiparallel RNA (red). Conditions:
PNA = DNA = 2.5 μM in 10 mM sodium phosphate buffer (pH 7.0)
and 100 mM NaCl at 20 °C.
Next, the DNA and RNA binding properties of mixed base
atfcPNA were investigated and compared with the correspond-
ing acpcPNA sequences (Table 2). Three sequences with
different G+C content were compared (%G+C content:
M10AT = 20; M10 = 40; and M10CG = 70). As shown in
Table 2, all atfcPNA sequences showed a strong DNA binding
affinity, and specifically in antiparallel orientation similar to
acpcPNA. They also exhibit very high sequence specificity as
shown by a large decrease in Tm values of mismatched compared
to the complementary hybrids (ΔTm ∼ 19−33 °C), which are in
the same range as acpcPNA (ΔTm ∼ 19−31 °C) and are much
more discriminating than aegPNA.11 It is interesting to note that
while acpcPNA·DNA hybrids did not show a definite relation-
ship between Tm and the base composition,10d the Tm of
atfcPNA showed a normal correlation with base composition
(i.e., the Tm is increased with %G+C content). Further studies
are clearly required in order to understand the basis of different
behaviors between the two PNA systems, but it is beyond the
scope of the present study.
In addition to DNA, atfcPNA can also bind to RNA in an
antiparallel fashion, albeit with much lower affinity compared to
the corresponding antiparallel DNA. Parallel hybrids with RNA
were observed in G+C-rich sequences for both atfcPNA and
acpcPNA, but the parallel hybrid was much less stable than the
antiparallel hybrid, especially in the case of atfcPNA. Moreover,
the RNA binding was highly sequence-specific as shown by a
large decrease of Tm value in the presence of mismatched RNA
targets. The normal correlation between Tm and base
composition was again observed in atfcPNA·RNA hybrids. In
addition, atfcPNA exhibits a greater selectivity for binding to
DNA over RNA than acpcPNA as shown by the larger Tm
difference between atfcPNA·RNA and atfcPNA·DNA hybrids
(−20.4, −16.5, and −14.6 °C for M10AT, M10, and M10CG,
respectively) compared to acpcPNA·RNA and acpcPNA·DNA
hybrids (−21.6, −11.0, and −6.5 °C for M10AT, M10, and
M10CG, respectively). Smaller differences were observed at
higher G+C content in both PNA systems. The results suggest
that atfcPNA and acpcPNA cannot effectively adjust themselves
to the more limited conformational space of RNA duplexes
exerted by the 2′-hydroxyl group in RNA, and that there are
subtle differences between structures of A+T-rich and G+C-rich
sequences.
nm and a very small negative band at 280 nm. The single-
stranded dA9 DNA showed two minima at 206 and 250 nm and
maxima at 220, 232 (shoulder), and 275 nm. Upon hybrid
formation, the negative band at 250 nm was markedly intensified
and shifted to a slightly shorter wavelength (248 nm).
Moreover, significant changes were observed in the nucleobase
absorption region (260−300 nm) as shown by the appearance
of a new negative band at 268 nm and a positive band at 285 nm.
These probably reflect a change in the orientation of the
nucleobase, most likely as a result of base−base pairing and
stacking in the atfcPNA·DNA duplex. The overall shape of the
CD spectrum of atfcPNA·DNA hybrid is quite similar to the
corresponding acpcPNA·DNA and DNA·DNA hybrid.10b,d It is
therefore reasonable to assume that these hybrids adopt similar
right-handed helical conformations. Heating caused the CD
signal to return to the sum of the single-stranded components
(Figure S23 of the Supporting Information). A plot of the CD
signal at 248 nm as a function of temperature gave a sigmoidal
curve (Figure S23 of the Supporting Information, inset). From
this CD melting curve, a CD Tm of around 60 °C was obtained
which is in good agreement to the Tm obtained by UV−vis
spectrophotometry.
In contrast to the homothymine sequence, CD spectra of
single-stranded mixed base atfcPNA suggested pronounced
intrastrand base stacking (Figure 1b, see also Figures S28−S30
of the Supporting Information). The CD spectra are remarkably
similar to single-stranded DNA as shown by a negative band at
205 nm, a strong positive band at 220 nm, a negative band at
256−260 nm, and a positive band around 280 nm with a
crossover point around 267−273 nm.23 Hybridization with
antiparallel DNA did not change the overall shape of the CD
spectra. However, the negative band at 260 nm became more
intense and slightly shifted toward a longer wavelength. These
are in agreement with tightening of the helical pitch after duplex
formation.14 The effect is most clearly observed in the G+C-rich
sequence M10CG. The overall shape of the CD spectra of
atfcPNA·DNA hybrids corresponds to the B-form of DNA·
DNA duplexes.24 On the other hand, CD spectra of atfcPNA·
RNA hybrids exhibited distinct features in the 250−300 nm
region (Figure 1b, see also Figures S28−S30 of the Supporting
Information). A positive band was observed between 245−256
nm together with a negative band with minima around 265 nm.
This suggests that the atfcPNA·DNA and atfcPNA·RNA hybrids
adopt different conformations, which could contribute to their
different stabilities.
Circular dichroism (CD) spectroscopy was also used to study
the binding between the T9 atfcPNA and DNA (Figure 1a).
The T9 atfcPNA exhibited weak CD signals in single-stranded
form. The only prominent features were the positive band at 210
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The shorter C−O bond as well as the presence of lone pair
electrons in atfcPNA resulted in reduced steric effects compared
to acpcPNA. It may also provide an additional potential
hydrogen-bonding site and repulsion with the negatively
charged phosphate group in the DNA/RNA strand. These
factors could potentially contribute to the subtle difference in
nucleic acid binding properties of atfcPNA and acpcPNA. An
NMR study of oligomers of the opposite enantiomer (2R,3R) of
trans-atfc suggested torsional angle (θ) values in the range of
52−64° based on coupling constant (3JH−H) analyses.13a These
values are much smaller than the optimal values obtained from
MD simulations of acpcPNA·DNA hybrids (99−102°).11 The
fact that atfcPNA can still form stable hybrids with DNA
suggests that the tetrahydrofuran ring should be sufficiently
flexible to adopt the required torsional angle. On the basis of
NMR experiments, no evidence of intramolecular hydrogen
bonding between the amide bonds and the oxygen atom of the
atfc ring was observed in trans-atfc oligomers.13a However, this
does not rule out the possibilities of interstrand hydrogen
bonding and/or additional interactions such as repulsion with
the phosphate group of DNA or RNA. The absence of detailed
three-dimensional structures of atfcPNA and its DNA/RNA
hybrids precludes us from drawing a more definite conclusion.
Solubility and Nonspecific Binding Properties of
atfcPNA versus acpcPNA. It was expected that the
replacement of the cyclopentane ring in acpcPNA with the
hydrophilic tetrahydrofuran ring in atfcPNA should result in a
less hydrophobic PNA backbone. The more polar nature of
atfcPNA is evidenced in the decreased retention time from
reverse-phase HPLC analyses (T9: atfcPNA 30.6 min, acpcPNA
33.3 min; M10: atfcPNA 25.1 min, acpcPNA 30.0 min) (Figures
S14, S19, S15, and S20 of the Supporting Information). Due to
the small synthesis scale, it was not possible to directly compare
the solubility because all lysine-modified PNA sequences are
readily soluble in water and saturation could not be achieved.
Acetylation of the C-terminal lysine modifier allowed the
comparison of solubility between T9 acpcPNA and atfcPNA.
Unfortunately, the solubility of T9 atfcPNA was only marginally
improved (425 21 μM or ca. 1.4 mg/mL) over T9 acpcPNA
(348 10 μM or ca. 1.1 mg/mL). This unexpected solubility
behavior might be interpreted in terms of a combination
between stable packing of the PNA molecules in the solid state
and unfavorable entropy of dissolution as proposed to
explaining the low aqueous solubility of cellulose25 and
cyclodextrins.26
Figure 2. Comparison of fluorescein-labeled acpcPNA (gray) and
atfcPNA (white) (M10Flu) for (a) nonspecific adsorption onto
polypropylene microcentrifuge tube and (b) self-aggregation as
demonstrated by fluorescence spectrophotometry.
atfcPNA signal does not change as much (95.8 3.6 and 105.8
1.4% of the original value for brands 1 and 2, respectively).
Reduction of the nonspecific self-aggregation of M10Flu
atfcPNA was further demonstrated by fluorescence spectrosco-
py (Figure 2b). At low concentrations (≤1 μM), both PNAs
showed comparable fluorescence. However, at higher concen-
trations, the fluorescence of M10Flu acpcPNA was significantly
lower and reached a plateau at concentrations above 5 μM due
to self-aggregation of the PNA, resulting in self-quenching of the
fluorescein label. The same leveling effect was also observed in
M10Flu atfcPNA but at twice as high a concentration.
Accordingly, at 10 μM, the fluorescence of M10Flu atfcPNA
is 70% brighter than acpcPNA at the same concentration.
Addition of complementary DNA to M10Flu acpcPNA and to
atfcPNA restored the fluorescence of both PNAs to the same
level due to repulsion between the negatively charged PNA·
DNA hybrids, which separated the fluorescein dyes from each
other. These results support the beneficial effects of the
hydrophilic ATFC spacer in reducing nonspecific interactions
of pyrrolidinyl PNA, which should further expand the
applications of these PNAs.
CONCLUSION
■
In conclusion, we report an efficient synthesis of Fmoc-
protected tetrahydrofuran cyclic-β-amino acid from 2-deoxy-D-
ribose. The cyclic amino acid was successfully incorporated as a
building block for synthesizing a new tetrahydrofuran-
containing pyrrolidinyl PNA (atfcPNA). This novel atfcPNA
retained the excellent DNA binding affinity and specificity of
pyrrolidinyl PNA, with improved selectivity toward hybrid-
ization to DNA over RNA, a fact that is interesting in its own
right and should deserve further investigation in the context of
development of a selective DNA binding agent. In addition, the
hydrophilic tetrahydrofuran backbone provided beneficial
effects in reducing nonspecific interactions and self-aggregation
generally observed in acpcPNA and most other PNA systems.
Similar to conventional PNA,14 nonspecific binding with
hydrophobic materials was occasionally observed with acpcP-
NA. This becomes a serious problem when the PNA is modified
with one or more hydrophobic dyes. To compare the
nonspecific binding behaviors of atfcPNA and acpcPNA, N-
terminal fluorescein-labeled atfcPNA and acpcPNA M10Flu
were synthesized and compared. Both fluorescein-labeled PNAs
form stable hybrids with their complementary DNA target with
Tm of 45.7 and 46.7 °C, respectively. The nonspecific adsorption
of the fluorescein-labeled PNAs by two different brands of
polypropylene plastic tubes were investigated (Figure 2a).
Fluorescence measurements of PNA solutions (50 nM) were
recorded before and after repeated transfer between the plastic
tube and the cuvette until there was no further change in the
fluorescence intensity. It was observed that the acpcPNA signal
decreased quite significantly (80.1 3.6 and 53.3 2.3% of the
original value for brands 1 and 2, respectively), while the
EXPERIMENTAL SECTION
■
General Methods. All chemicals were purchased from commercial
sources and were used as received without further purification. Solvents
were dried over CaH2 or 4 Å molecular sieves and then freshly distilled
under argon prior to use. IR spectra were acquired on a FT-IR
spectrometer using either attenuated total reflection (ATR) or
transmission module. Optical rotations ([α]D) were measured at the
specified temperature using sodium light (D line, 589.3 nm);
concentrations (c) are reported as g/100 mL. 1H and 13C NMR
spectra were recorded at 400 and 100 MHz, respectively. High-
7062
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Article
resolution mass spectra (HRMS) were recorded in electrospray
ionization-time-of-flight (ESI-TOF) mode. MALDI-TOF mass spectra
of all atfcPNAs were obtained in linear positive ion mode using α-
cyano-4-hydroxy cinnamic acid (CCA) as a matrix.
were dried over Na2SO4 and evaporated. The crude product (2.31 g)
was purified by column chromatography to afford compound 6 as a
colorless oil (1.92 g, 4.92 mmol, 86%). [α]D23 +30° (c 1.0, CH2Cl2). IR
(thin film): 2958.2, 2880.1, 1716.8, 1606.8, 1447.6, 1363.7, 1268.2,
1172.7, 1091.6, 1016.4, 955.6, 906.4, 816.7, 753.0, 669.1, 553.4 cm−1.
1H NMR (400 MHz, CDCl3): δ 7.87 (d, J = 8.3 Hz, 2H), 7.77 (d, J =
8.1 Hz, 2H), 7.28 (d, J = 8.2 Hz, 2H), 7.24 (d, J = 8.1 Hz, 2H), 5.00 (m,
1H), 4.15−4.25 (m, 3H), 4.05 (m, 1H), 3.94 (m, 1H), 2.42 (s, 3H),
2.40 (s, 3H), 2.15−2.20 (m, 2H). 13C NMR (100 MHz, CDCl3): δ
166.1, 145.1, 144.0, 133.6, 130.0, 129.7, 129.2, 127.8, 126.9, 82.2, 81.6,
67.4, 63.6, 33.1, 21.7, 21.6. HRMS (ESI+): m/z calcd for C20H22O6SNa
[M + Na+], 413.1029; found, 413.1026.
[(2R,3R)-3-Hydroxytetrahydrofuran-2-yl]methyl-4-methylben-
zoate (7). A mixture of compound 6 (1.08 g, 2.77 mmol) and NaNO2
(0.96 g, 13.9 mmol) in DMSO (10 mL) was heated to 120 °C for 5 h.
After completion as monitored by TLC analysis (hexanes:EtOAc 1:1;
Rf = 0.27), the reaction was diluted with water. The aqueous layer was
then extracted with CH2Cl2 (3 × 10 mL) and the combined organic
extracts were washed with brine, dried over Na2SO4, and evaporated.
The crude product was purified by column chromatography to afford a
mixture of 7 and the toluoyl migrate product 7′ in a 1:1 ratio as
determined by NMR analysis (0.41 g, 1.75 mmol, combined yield
63%). When the reaction time was reduced to 2 h at 120 °C, the
product was obtained as 4.5:1 regioisomeric mixture of product 7:7′ in
58% combined yield.
Methyl 2-Deoxy-D-ribofuranoside (3) (Mixture of α and β
Anomers). Methyl 2-deoxy-D-ribofuranoside 3 was synthesized
according to the literature procedure with a slight modification on
the catalyst quantity and reaction time.16,17 2-Deoxy-D-ribose (11.4 g,
85.0 mmol) was dissolved in anhydrous methanol (55 mL) under a
nitrogen atmosphere, and then methanesulfonic acid (0.27 mL, 4.16
mmol) was added at room temperature. The reaction reached
completion after stirring for 30 min at room temperature as confirmed
by TLC analysis (CH2Cl2:MeOH 9:1, p-anisaldehyde stain; Rf = 0.42).
The reaction was then quenched by portionwise addition of 4-(N,N-
dimethylamino)pyridine (DMAP) (1.02 g, 8.35 mmol). The reaction
mixture was concentrated under reduced pressure by azeotropic
distillation with toluene to afford a brown syrup (14.15 g, >90% purity
by TLC), which was used for the next steps without further purification.
Methyl 5-O-p-toluoyl-2-deoxy-D-ribofuranoside (4) (Mixture of α
and β Anomers). p-Toluoyl chloride (13 mL, 98.3 mmol) dissolved in
anhydrous CH2Cl2 (20 mL) was added dropwise to a solution of crude
3 (prepared from 85.0 mmol of 2) in 1:3 anhydrous pyridine/CH2Cl2
(80 mL) at 0 °C and stirred for 5 h. The reaction mixture was then
diluted with CH2Cl2 (20 mL) and washed with cold H2O (2 × 50 mL)
and 5% aqueous CuSO4. The combined organic extracts were washed
with saturated NaHCO3 (2 × 50 mL), followed by 2 M HCl (2 × 50
mL), and then dried over Na2SO4 and concentrated under reduced
pressure to afford a brown syrup (21.64 g). The crude product was
purified by column chromatography on silica gel to yield compound 4
as a colorless oil (15.1 g, 56.8 mmol, 67% yield from 2). TLC analysis
For characterization purposes, the 1:1 regioisomeric mixture from
the experiment above (0.41 g, 1.75 mmol) was dissolved in anhydrous
CH2Cl2 (3 mL), cooled to 0 °C, and then treated with Et3N (1.25 mL,
8.96 mmol) and Bz2O (0.3370 g, 1.49 mmol). The reaction mixture was
concentrated to dryness and purified by column chromatography to
yield 7 as colorless oil (0.18 g, 0.76 mmol, 28% overall yield from 6).
This corresponds to a 89% recovery from the mixture. Mp: 65−67 °C;
24
(hexanes:EtOAc 3:2; p-anisaldehyde stain; Rf = 0.27). [α]D +67° (c
0.5, CHCl3). IR (thin film): 3467.5, 2949.5, 2917.7, 2828.0, 1716.7,
1
1609.7, 1447.6, 1268.2, 1181.4, 1077.2, 842.8, 758.8 cm−1. H NMR
23
[α]D −4° (c 1, CH2Cl2). IR (ATR) 3420.7, 2986.2, 2950.6, 2923.3,
(400 MHz, CDCl3) (major isomer): δ 7.92 (d, J = 8.2 Hz, 2H), 7.25 (d,
J = 8.1 Hz, 4H), 5.16 (m, 1H), 4.32−4.41 (m, 3H), 4.26 (m, 1H), 3.42
(s, 3H), 3.01 (m, 1H), 2.43 (s, 3H), 2.20 (m, 1H), 2.08 (m, 1H). 13C
NMR (100 MHz, CDCl3) (major isomer): δ 166.4, 143.9, 129.7, 129.1,
127.0, 105.5, 85.1, 73.1, 64.4, 54.9, 40.9, 21.6. HRMS (ESI+): m/z calcd
for C14H18O5Na [M + Na+], 289.1052; found, 289.1061.
2896.0, 2868.7, 1704.4, 1611.5, 1444.7, 1269.8, 1174.2, 1100.4, 1021.1,
969.2, 834.5, 750.6, 693.2 cm−1. 1H NMR (400 MHz, CDCl3): δ 7.94
(d, J = 8.2 Hz, 2H), 7.24 (d, J = 8.0 Hz, 2H), 4.77 (dd, J = 7.1, 11.6 Hz,
1H), 4.34−4.39 (m, 2H), 4.11 (m, 1H), 3.96 (ddd, J = 3.3, 5.4, 7.1 Hz,
1H), 3.89 (dt, J = 3.8, 8.6 Hz, 1H), 2.41 (s, 3H), 2.18 (m, 1H), 2.05 (m,
1H). 13C NMR (100 MHz, CDCl3): δ 167.2, 144.1, 129.8, 129.2, 126.9,
80.6, 71.5, 66.5, 62.6, 35.1, 21.7. HRMS (ESI+): m/z calcd for
C13H16O4Na [M + Na+], 259.0946; found, 259.0941.
Methyl 5-O-p-toluoyl-3-O-tosyl-2-deoxy-D-ribofuranoside (5). A
solution of 4 (4.26 g, 16.0 mmol) and DMAP (183 mg, 1.5 mmol) in
anhydrous CH2Cl2 (10 mL) was cooled to 0 °C under a N2 atmosphere
and then treated with Et3N (5.00 mL, 35.8 mmol) and a solution of
TsCl (4.011 g, 21.0 mmol) in CH2Cl2 (5 mL). The reaction mixture
was allowed to warm to room temperature and monitored by TLC
(hexanes:EtOAc 3:2; p-anisaldehyde stain; Rf = 0.42). After
completion, the reaction mixture was quenched at 0 °C with 2 M
HCl, neutralized with saturated aqueous NaHCO3 and extracted with
EtOAc. The combined organic extracts were washed with water (40
mL), brine (40 mL), dried over Na2SO4, and evaporated. The crude
product (6.40 g) was purified by column chromatography on silica gel
[(2R,3R)-3-(Methylsulfonyloxy)tetrahydrofuran-2-yl]methyl 4-
methylbenzoate (8). A solution of 7 (0.49 g, 2.07 mmol) in anhydrous
CH2Cl2 (4 mL) was cooled to 0 °C and treated with DMAP (50 mg,
0.41 mmol), Et3N (1.15 mL, 8.25 mmol), and MsCl (0.48 mL, 6.20
mmol). After 1 h, the reaction was complete as indicated by TLC
(CH2Cl2:acetone 4:1; Rf = 0.76) and was diluted with H2O (10 mL)
and EtOAc (10 mL). The aqueous layer was extracted with EtOAc (3 ×
10 mL), and the combined organic extracts were washed with brine,
dried over Na2SO4, and concentrated under reduced pressure. The
crude product (0.62 g) was purified by column chromatography to
afford 8 as a white solid (0.58 g, 1.85 mmol, 90%). Mp: 102−104 °C.
[α]D23 −44° (c 1.0, CH2Cl2). IR (KBr): 3424.1, 3024.8, 2964.0, 2940.8,
2891.6, 2862.7, 1722.5, 1609.6, 1349.2, 1271.1, 1178.5, 1109.0, 1010.6,
967.2, 897.7, 750.2, 524.4 cm−1. 1H NMR (400 MHz, CDCl3): 7.94 (d,
J = 7.8 Hz, 2H), 7.24 (d, J = 8.1 Hz, 2H), 5.38 (m, 1H), 4.53 (m, 2H),
4.25 (dt, J = 4.0, 6.1 Hz, 1H), 4.15 (m, 1H), 3.96 (dt, J = 2.7, 8.5 Hz,
1H), 3.01 (s, 3H), 2.41 (s, 3H), 2.39 (m, 2H). 13C NMR (100 MHz,
CDCl3): δ 166.2, 144.0, 129.8, 129.2, 127.0, 79.9, 78.8, 66.5, 62.2, 38.5,
34.0, 21.7; HRMS (ESI+): m/z calcd for C14H18O6SNa [M + Na+],
337.0722; found, 337.0716.
24
to afford compound 5 (5.59 g, 13.3 mmol, 83%). [α]D +75° (c 0.5,
CHCl3). IR (thin film): 3027.6, 2949.5, 2929.3, 2836.7, 1922.2, 1716.7,
1606.8, 1450.5, 1363.7, 1276.8, 1172.7, 1106.1, 1056.9, 984.6, 920.9,
1
848.5, 810.9, 753.0, 666.2, 567.8, 550.5 cm−1. H NMR (400 MHz,
CDCl3): δ 7.85 (d, J = 8.0 Hz, 2H), 7.77 (d, J = 8.3 Hz, 2H), 7.27 (d, J =
8.3 Hz, 2H), 7.24 (d, J = 8.0 Hz, 2H), 5.06 (m, 1H), 4.93 (m, 1H),
4.38−4.44 (m, 2H), 4.20 (m, 1H), 3.36 (s, 3H), 2.42 (s, 3H), 2.39 (s,
3H), 2.34 (m, 1H), 2.13 (m, 1H). 13C NMR (100 MHz, CDCl3): δ
166.0, 145.1, 144.0, 133.4, 129.9, 129.7, 129.1, 127.9, 126.9, 104.5, 80.1,
79.4, 63.0, 55.1, 39.2, 21.7, 21.6. HRMS (ESI+): m/z calcd for
C21H24O7SNa [M + Na+], 443.1135; found, 443.1137.
5-O-p-toluoyl-3-O-tosyl-1,2-dideoxy-D-ribose (6). A solution of
compound 5 (2.41 g, 5.73 mmol) in anhydrous CH2Cl2 (2 mL) was
cooled to 0 °C and treated with Et3SiH (2.90 mL, 18.2 mmol), followed
by dropwise addition of BF3·OEt2 (2.00 mL, 16.2 mmol). The progress
of the reaction was monitored by TLC (Hexanes:EtOAc 1:1; negative
p-anisaldehyde stain; Rf = 0.58). After completion, the reaction mixture
was neutralized with saturated aqueous NaHCO3 (30 mL) and
extracted with CH2Cl2 (3 × 10 mL). The combined organic extracts
[(2S,3S)-3-Azidotetrahydrofuran-2-yl]methyl-4-methylbenzoate
(9). A solution of 8 (0.14 g, 0.46 mmol) in DMSO (2 mL) was treated
with NaN3 (0.21 g, 3.15 mmol) and then heated at 90 °C for 2 h. After
the reaction reached completion as monitored by TLC analysis
(hexanes:EtOAc 3:2; Rf = 0.62), it was diluted with water (15 mL) and
extracted with EtOAc (3 × 15 mL). The combined organic extracts
were dried over Na2SO4 and concentrated to dryness to afford
compound 9 as a colorless oil (113 mg, 0.43 mmol, 95%), which was
23
subjected to the next step without further purification. [α]D +64° (c
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Article
1.6, CH2Cl2). IR (thin film): 2952.4, 2872.4, 2098.7, 1716.7, 1606.7,
homogeneous solution. After stirring overnight, the solution was
diluted with water and extracted with diethyl ether. The aqueous phase
was acidified to pH 2 with concentrated HCl to give a white suspension.
After filtration, the precipitate was air-dried to afford compound 13 as a
white solid (0.28 g, 0.79 mmol, 87%). Mp: 166−168 °C. [α]D23 +42° (c
1.0, DMSO). IR (KBr): 3308.4, 3062.4, 2946.6, 2362.1, 2341.8, 1722.5,
1
1271.1, 1172.7, 1103.2, 750.2 cm−1. H NMR (400 MHz, CDCl3): δ
7.93 (d, J = 8.0 Hz, 2H), 7.25 (d, J = 8.0 Hz, 2H), 4.38 (m, 2H), 4.03−
4.12 (m, 3H), 3.95 (m, 1H), 2.42 (s, 3H), 2.29 (m, 1H), 2.08 (m, 1H).
13C NMR (100 MHz, CDCl3): δ 166.3, 144.0, 129.7, 129.2, 127.0, 81.5,
67.4, 64.4, 62.9, 32.2, 21.7. HRMS (ESI+): m/z calcd for
1
1690.7, 1540.2, 1447.6, 1259.6, 1099.4, 732.8 cm−1. H NMR (400
C13H15N3O3Na [M + Na+], 284.1006; found, 284.1003.
MHz, DMSO-d6): δ 12.75 (br s, 1H), 7.89 (d, J = 7.4 Hz, 2H), 7.74 (d,
J = 7.5 Hz, 1H), 7.69 (m, 2H), 7.42 (t, J = 7.0 Hz, 2H), 7.34 (t, J = 7.0
Hz, 2H), 4.33 (d, J = 6.4 Hz, 2H), 4.22 (t, J = 6.9 Hz, 1H), 4.17 (m,
1H), 4.09 (m, 1H), 3.90 (t, J = 6.8 Hz, 2H), 2.09 (m, 1H), 1.79 (m,
1H). 13C NMR (100 MHz, DMSO-d6): δ 172.8, 155.5, 143.7, 140.6,
127.5, 127.0, 125.0, 120.0, 81.1, 67.0, 65.3, 55.3, 46.6, 31.8. HRMS (ESI
+): m/z calcd for C20H19NO5Na [M + Na+], 376.1155; found,
376.1180.
[(2S,3S)-3-(tert-Butoxycarbonylamino)tetrahydrofuran-2-yl]-
methyl 4-methylbenzoate (10). A solution of 9 (0.47 g, 1.8 mmol) in
MeOH (5 mL) was treated with Boc2O (1.03 g, 4.72 mmol) and Pd/C
(20% w/w, 74.2 mg). The reaction flask was flushed with H2 gas and a
H2 balloon was attached through a rubber septum. The reaction was
monitored by TLC analysis (hexanes:EtOAc 3:2; ninhydrin stain; Rf =
0.46) until it reached completion. It was then filtered through Celite,
concentrated to dryness, and the residue was washed with hexanes to
afford 10 as a white solid (0.43 g, 1.28 mmol, 71%). Mp: 108−110 °C.
[α]D23 +28° (c 1.0, CH2Cl2). IR (KBr): 3348.9, 2975.6, 2937.9, 2868.5,
1724.8, 1682.0, 1609.0, 1528.6, 1432.5, 1369.5, 1271.1, 1172.7, 1108.3,
1077.2, 1021.5, 1001.2, 882.6, 884.9, 753.1, 607.6 cm−1. 1H NMR (400
MHz, CDCl3): δ 7.93 (d, J = 8.2 Hz, 2H), 7.22 (d, J = 8.1 Hz, 2H), 4.71
(br s, 1H), 4.49 (dd, J = 3.5, 11.7 Hz, 1H), 4.34 (dd, J = 5.9, 11.8 Hz,
1H), 4.16 (m, 1H), 3.92−4.06 (m, 3H), 2.40 (s, 3H), 2.34 (m, 1H),
1.83 (dt, J = 5.7, 12.8 Hz, 1H), 1.44 (s, 9H). 13C NMR (100 MHz,
CDCl3): δ 166.5, 155.5, 143.7, 129.8, 129.1, 127.2, 82.9, 79.9, 67.2,
65.1, 53.2, 33.3, 28.4, 21.6. HRMS (ESI+): m/z calcd for
C18H25NO5Na [M + Na+], 358.1625; found, 358.1635.
Pentafluorophenyl (2S,3S)-3-{[(9H-fluoren-9-yl)methoxy]-
carbonylamino}tetrahydrofuran-2-carboxylate (1). A suspension of
compound 13 (0.22 g, 0.62 mmol) in CH2Cl2 (2 mL) was treated with
DIEA (600 μL, 3.45 mmol) and PfpOTfa (600 μL, 3.39 mmol) in three
portions. The completion of the reaction was monitored by TLC
analysis (hexanes:EtOAc 1:1; Rf = 0.50). The reaction mixture was
diluted with CH2Cl2 (10 mL), washed with 1 M HCl and saturated
aqueous NaHCO3 and dried over anhydrous Na2SO4. After removal of
the solvent, the resulting pink oil was sonicated with hexanes to give a
white suspension which was collected by filtration to give 1 as a white
solid (0.207 g, 0.40 mmol, 65%). Mp: 123−125 °C. [α]D23 +57° (c 1.0,
CH2Cl2). IR (ATR): 3309.4, 1799.7, 1687.6, 1550.0, 1519.6, 1291.0,
1234.9, 1066.9, 1031.9, 994.6, 761.2, 735.6, 665.6. 1H NMR (400 MHz,
CDCl3): δ 7.77 (d, J = 7.4 Hz, 2H), 7.58 (d, J = 7.2 Hz, 2H), 7.41 (t, J =
6.9 Hz, 2H), 7.31 (t, J = 7.1 Hz, 2H), 5.06 (d, J = 5.3, 1H), 4.70 (m,
1H), 4.62 (m, 1H), 4.50 (m, 2H), 4.19 (m, 2H), 4.12 (m, 1H), 2.36 (m,
1H), 1.98 (m, 1H). 13C NMR (100 MHz, CDCl3): δ 167.3, 155.5,
143.7, 141.4, 127.8, 127.0, 124.9, 120.0, 81.7, 68.3, 66.9, 56.2, 47.3, 31.9.
19F NMR (376 MHz, CDCl3): δ −161.8 (t, J = 20.2 Hz), −157.1 (t, J =
tert-Butyl (2S,3S)-2-(Hydroxymethyl)tetrahydrofuran-3-ylcarba-
mate (11). A mixture of 10 (0.39 g, 1.16 mmol) and LiOH·H2O
(0.13 g, 3.1 mmol) was dissolved in 1:1 THF:H2O (4 mL) and stirred
at room temperature overnight. The completion of the reaction was
confirmed by TLC analysis (EtOAc:hexanes 4:1; ninhydrin stain; Rf =
0.33), and then the organic solvent was removed by rotary evaporation,
and the aqueous residue was extracted with CH2Cl2 (3 × 10 mL). The
combined organic phases were dried over anhydrous Na2SO4 and
evaporated to give compound 11 as a white solid (0.23 g, 1.06 mmol,
19.9 Hz), −152.4 (d, J = 18.0 Hz). HRMS (ESI+) m/z calcd for
23
1
C26H18F5NO4K [M + K+], 542.0788; found, 542.0795.
91%). Mp: 85−87 °C. [α]D −11° (c 1.0, CH2Cl2). H NMR (400
MHz, CDCl3): δ 4.47 (s, 1H), 3.85−4.00 (m, 3H), 3.65−3.69 (m, 3H),
2.87 (s, 1H), 2.29 (m, 1H), 1.79 (m, 1H), 1.44 (s, 9H). 13C NMR (100
MHz, CDCl3): δ 155.9, 85.1, 80.2, 66.7, 63.1, 52.9, 33.1, 28.3. IR
(ATR): 3346.8, 2987.4, 2964.1, 2908.1, 2849.5, 1676.0, 1522.0, 1363.3,
1300.3, 1239.6, 1160.3, 1097.3, 1073.9, 1015.6, 866.3, 621.2 cm−1.
HRMS (ESI+): m/z calcd for C10H19NO4Na [M + Na+], 240.1206;
found, 240.1207.
Synthesis of atfcPNA. All atfcPNA were manually synthesized at
1.5 μmol scale on a Tentagel S-RAM resin (0.24 mmol/g loading) from
the four Fmoc-protected pyrrolidinyl PNA monomers (ABz, T, CBz, and
G
Ibu) and pentafluorophenyl-activated ATFC spacer 1 following the
previously published protocol for acpcPNA synthesis.10b,d The
nucleobase protecting groups (Bz, Ibu) were removed by treating the
resin with 1:1 aqueous ammonia:dioxane at 60 °C overnight. The crude
PNA was obtained by TFA cleavage and purified by reverse-phase
HPLC (C18 column, 0.1% TFA in water−methanol gradient).
Fluorescence Spectroscopy to Compare Nonspecific Inter-
actions and Self-Aggregation of acpcPNA and atfcPNA. (a)
Nonspecific adsorption of fluorescein-modified acpcPNA and atfcPNA
M10Flu were compared by fluorescence measurements. The PNA
samples were prepared at 0.05 μM in 10 mM sodium phosphate buffer
pH 7.0 (1000 μL) at 25 °C. The solution was transferred back and forth
between the cuvette and a 1.5 mL polypropylene microcentrifuge tube,
and the fluorescence spectra were recorded after each transfer until no
further change was observed. The average fluorescence at 520 nm and
standard deviation were calculated from the last three measurements
after the signal was constant. The excitation wavelength was 480 nm,
slit widths 5 nm, and the PMT voltage was set to high. (b) Self-
aggregation of fluorescein-modified acpcPNA and atfcPNA M10Flu
were compared by fluorescence measurements. The fluorescence data
was plotted against concentration in the range of 1 to 10 μM or until a
plateau was reached. The excitation wavelength was 480 nm, slit widths
5 nm, and the PMT voltage was set to 500.
(2S,3S)-3-(tert-Butoxycarbonylamino)tetrahydrofuran-2-carbox-
ylic acid (12). To a solution of 11 (0.27 g, 1.24 mmol) in 1:1
acetonitrile:H2O was added TEMPO (95 mg, 0.61 mmol) and
bis(acetoxy)iodobenzene (BAIB) (1.13 g, 3.5 mmol). The reaction
mixture was stirred overnight at room temperature. The completion of
the reaction was monitored by TLC analysis (EtOAc; ninhydrin stain;
Rf = 0.17). The pH of the resulting solution was adjusted 8−9 by
addition of solid NaHCO3 and extracted with EtOAc. The collected
aqueous phase was acidified to pH 2 with solid NaHSO4 followed by
extraction with EtOAc. The combined organic phases were evaporated
22
to dryness to give 12 as a colorless oil (0.27 g, 1.2 mmol, 94%). [α]D
+49° (c 1.5, DMSO). IR (thin film): 3328.6, 2972.7, 1710.9, 1528.6,
1
1363.7, 1279.7, 1161.1, 1100.3, 1016.4 cm−1. H NMR (400 MHz,
DMSO-d6): δ 12.60 (br s, 1H), 7.27 (d, J = 6.9 Hz, 1H), 4.10 (m, 1H),
4.04 (m, 1H), 3.83−3.92 (m, 2H), 2.06 (m, 1H), 1.76 (dt, J = 6.3, 12.0
Hz, 1H), 1.38 (s, 9H). 13C NMR (100 MHz, DMSO-d6): δ 172.7,
154.9, 81.0, 77.9, 67.1, 54.9, 31.6, 28.1. HRMS (ESI+): m/z calcd for
C10H17NO5Na [M + Na+], 254.0999; found, 254.0992.
(2S,3S)-3-{[(9H-Fluoren-9-yl)methoxy]carbonylamino}tetra-
hydrofuran-2-carboxylic acid (13). Compound 12 (0.21 g, 0.91
mmol) was dissolved in 1:1 TFA:CH2Cl2 (6 mL) and stirred at room
temperature for 30 min. The solvent was removed by flushing with N2.
The crude amino acid was dissolved in H2O (4 mL), and the pH of the
solution was adjusted to 8−9 by the addition of solid NaHCO3. Next,
FmocOSu (0.31 g, 0.92 mmol) dissolved in 0.5 mL of DMSO was
added in small portions along with more NaHCO3 to maintain the pH
around 8−9. To this mixture, MeCN was added to make a
ASSOCIATED CONTENT
■
S
* Supporting Information
1H and 13C NMR spectra for numbered compounds, HPLC
chromatogram, and MALDI-TOF mass spectra of PNA, as well
as additional spectroscopic data. The Supporting Information is
7064
DOI: 10.1021/acs.joc.5b00890
J. Org. Chem. 2015, 80, 7058−7065

The Journal of Organic Chemistry
Article
Analogues; Merino, P., Ed.; Wiley-VCH: New Jersey, 2013; pp 847−
880.
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available free of charge on the ACS Publications website at DOI:
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AUTHOR INFORMATION
■
Corresponding Author
*E-mail: vtirayut@chula.ac.th.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
PNA & XNA 2011, 2, 50−59. (e) Mansawat, W.; Vilaivan, C.; Balaz
́
s,
Financial support of this work provided by the Thailand
Research Fund (DPG5780002) and the Thai government
stimulus package 2 (TKK2555, SP2) under the Project for
Establishment of Comprehensive Center for Innovative Food,
Health Products and Agriculture is gratefully acknowledged. We
thank Haruthai Pansuwan and Nattawut Yotapan for partial data
collection.
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