Clickable Cγ-azido(methylene/butylene) peptide nucleic acids and their clicked fluorescent derivatives: Synthesis, DNA hybridization properties, and cell penetration studies

Article abstract of DOI:10.1021/jo500834u

Synthesis, characterization, and DNA complementation studies of clickable C^γ-substituted methylene (azm)/butylene (azb) azido PNAs show that these analogues enhance the stability of the derived PNA:DNA duplexes. The fluorescent PNA oligomers synthesized by their click reaction with propyne carboxyfluorescein are seen to accumulate around the nuclear membrane in 3T3 cells.

Full text of DOI:10.1021/jo500834u

Note
pubs.acs.org/joc
Clickable Cγ‑Azido(methylene/butylene) Peptide Nucleic Acids and
Their Clicked Fluorescent Derivatives: Synthesis, DNA Hybridization
Properties, and Cell Penetration Studies
Deepak R. Jain and Krishna N. Ganesh*
Chemical Biology Unit, Indian Institute of Science Education and Research, Dr Homi Bhabha Road, Pune 411008, India
S
* Supporting Information
ABSTRACT: Synthesis, characterization, and DNA comple-
mentation studies of clickable Cγ-substituted methylene
(azm)/butylene (azb) azido PNAs show that these analogues
enhance the stability of the derived PNA:DNA duplexes. The
fluorescent PNA oligomers synthesized by their click reaction
with propyne carboxyfluorescein are seen to accumulate
around the nuclear membrane in 3T3 cells.
moiety have been employed to synthesize triazolyl PNA
oligomers by click reaction on solid phase.20
eptide nucleic acids are effective DNA analogues in which
Pthe sugar phosphate backbone is replaced with a
pseudopeptide backbone in the form of achiral 2-(aminoethyl)-
glycine (aeg) linkage (Figure 1a). Nucleobases (A/T/G/C) are
attached through a methylene carbonyl linker to this backbone
at the amino nitrogen.1,2 PNA hybridizes with complementary
DNA/RNA via Watson−Crick base pairing with higher avidity
compared to DNA:DNA or DNA:RNA duplexes which may
arise from a high flexibility and the absence of charge in the
backbone. PNAs are resistant to both proteases and nucleases
and consequently have a longer life span in the cellular
environment.3 The simplicity of chemical structure, ease of
synthesi,s and superior binding properties have made PNA an
attractive gene regulatory agent.4−6 However, these suffer from
poor aqueous solubility, cell penetrability, and equal affinity for
binding to cDNA and RNA, which reduces their target
specificity by half.7−9 Many PNA modifications have been
rationally designed to address these concerns. They include
modifications in the backbone to preorganize PNA con-
formation to tune with that of cDNA or RNA, introduction of
charged moieties to enhance solubility, and conjugation with
transfer molecules to improve cell penetrability.10−12 Another
approach is to make them inherently cationic and chiral to
simultaneously address these issues.13 Employing click
chemistry, PNAs carrying N-terminus azide function can be
conjugated with application specific functional ligands such as
ferrocene (electrochemical detection),14a 2,2′-dipicolylamine
Figure 1. Chemical structures of (a) aeg PNA, (b) γ(R)-azm aeg PNA,
and (c) γ(S)-azb aeg PNA.
Here we describe an approach to synthesize site-specific
azido PNAs (Figure 1b,c) wherein the clickable azido moiety is
placed on the PNA chain, separated from backbone by
methylene or buytlene chain to examine the spacer dependent
effects on the DNA complementation. Employing click
chemistry, fluorophore carboxyfluorescein is conjugated to
these PNAs. The synthesis of target azido PNA monomers
(11a and 11b), their insertion at specific sites into a PNA
sequence to give azido PNAs, hybridization with comple-
mentary and mismatch DNA, and click attachment of
carboxyfluorescein PNAs for live cell imaging are reported. It
is shown that both methylene- and butylene-linked azido PNAs
show improved efficiency of hybridization with cDNA
compared to unmodified PNA and the butylene-linked azido
PNAs perform better than the methylene PNAs.
(
99mTc-labeling, biodistribution),14b quinoline (Re complex,
bioimaging),14c or fluorophores (cell penetration13 and
molecular beacons15) or use in addressable PNA micro-
arrays.16,17 Site-specific labeling via click chemistry has been
achieved via either azide bearing nucleobase18 or at pyrrolidine
ring within the backbone.19 PNA monomers bearing azide
Received: April 22, 2014
Published: June 18, 2014
© 2014 American Chemical Society
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Note
Scheme 1. Synthesis of γ-azm aeg PNA and γ-azb aeg PNA Monomers
a
Table 1. PNA Sequences and UV−Tmof PNA/DNA Duplexes
a
See the Supporting Information for mass spectra; T = azidomethylene (azm) PNA unit and T = azidobutylene (azb) PNA unit; Tf and Tf are the
carboxyfluorescein derivatives; DNA 1, 5′ACTGAGGTAA3′(ap, antiparallel); DNA 2, 5′ACTGCGGTAA3′ (mm, mismatch); [PNA/DNA] = 2
μM each strand. Buffer: sodium phosphate (10 mM), NaCl (10 mM) and EDTA (0.1 mM); nb = not binding.
SYNTHESIS OF Cγ-AZIDOALKYLIDENE PNA
MONOMERS
mesylate 6a that was immediately reacted with potassium
■
phthalimide to obtain the N-protected compound 7a.
Deprotection of phthalimide with hydrazine hydrate gave the
free amine which was in situ N-alkylated with ethyl
bromoacetate to yield product 8a. The N-acylation of this
compound with chloroacetyl chloride led to the chloromethyl
compound 9a, and subsequent reaction with nucleobase
thymine gave γ-(azidomethylene)aminoethylglycyl ethyl ester
10a in quantitative yield. The γ-azm aeg PNA ester 10a was
hydrolyzed using aqueous lithium hydroxide to obtain the γ-
azm aeg PNA acid 11a.
The synthesis of γ-azb aeg PNA-T monomer was achieved
starting from N-α-Boc-L-lysine (2b) that was converted to the
azido derivative 3b and then following a similar reaction
sequence as for 11a gave the γ-azidobutylene aeg PNA acid 11b.
All the intermediate new compounds and the final monomers
were characterized by analytical and spectroscopic data
(Supporting Information).
The synthesis of desired Cγ-azidomethylene (11a) and Cγ-
azidobutylene (11b) PNA-T monomers containing thymine
nucleobase was started from the commercially available N-α-
Boc-L-asparagine (1) (Scheme 1) for methylene spacer and N-
α-Boc-L-lysine (2b) for butylene spacer. The side chain amido
group in compound 1 was converted to the amino function via
Hofmann rearrangement which yielded 3-amino-2-(Boc-
amino)propanoic acid (2a).21 This was subjected to diazo
transfer reaction for the conversion of primary amine to azide
derivative. Triflyl azide synthesized as per known method22 was
reacted with amine 2a in the presence of CuSO4·5H2O and
triethylamine to achieve azo transfer to the azide 3a. This was
esterified using dimethyl sulfate/activated K2CO3 to give the
methyl ester 4a that was reduced with NaBH4 to the primary
alcohol 5a. It was treated with mesyl chloride to give the
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Note
SOLID-PHASE SYNTHESIS OF PNA OLIGOMERS
AND THEIR CHARACTERIZATION
■
The PNA oligomers were synthesized by manual solid-phase
peptide synthesis on L-lysine-derivatized MBHA resin using the
Boc-chemistry protocol23 and standard HOBt−HBTU coupling
reagents. The PNA oligomers (Table 1) incorporating
azidoalkylidene units at chosen sites in the unmodified PNA
sequence 1 were obtained by coupling with the monomers 11a
and 11b at the desired positions of the PNA sequence. The
azido PNAs (PNA 2−9) were transformed to the fluorescently
labeled PNA oligomers (PNA 10−16) by on-resin click
reaction with alkyne derivative of 5(6)-carboxyfluorescein24 in
the presence of copper (Supporting Information). This enabled
attachment of a fluorophore at any desired position in the
sequence. At the end of the assembly, the PNA oligomers were
cleaved from the resin by TFA−TFMSA.25 at which time the
Cbz-protection on exocyclic amine of nucleobases was also
simultaneously removed to give crude PNAs. All PNA
oligomers were purified by reversed-phase HPLC and
characterized by MALDI-TOF spectrometry (Supporting
Information). The various modified and unmodified aeg PNA
oligomers which were synthesized are listed in Table 1. The
fluorescent PNAs azm-T(cf) PNA 10−13 and azb-T(cf) PNA
14−16 obtained from click reactions were characterized by
their fluorescence emission band at 520 nm upon excitation at
484 nm arising from 5(6)-carboxyfluorescein moiety (Support-
ing Information).
Figure 2. Comparative ΔTmvalues for PNA:DNA duplexes of γ-
azm(PNA 2−5) and γ-azb(PNA 6−9) with cDNA 1 and mismatch
DNA 2. ΔTm (green bars):stabilization compared to aeg-PNA 1:DNA
1 duplex. ΔTm(red bars):destabilization of PNA:DNA 2 duplexes
compared to PNA:DNA 1 duplexes.
thermal stability of azidoalkylidene PNA:DNA hybrids suggest
that (i) the extent of stability depends on the number of
methylenes in the side chain, with PNAs having longer butyl
chain showing better stability than those shorter methylene
chains and (ii) the stabilizing factors are site dependent, but not
additive in nature.
Fluorescently labeled PNAs generated from online click
reaction of various azido PNAs with propyne carboxyfluor-
escein were examined for their abilities to form duplexes with
cDNA 1 and mismatch DNA 2. The extent of relative
stabilization/destabilization compared to their azido analogues
are shown in Table 1 (see the Supporting Information). The
azm PNAs (PNA 10−13) showed destabilization (ΔTm= −3.4
to +0.4 °C) at almost all sites, while the azb analogues were
slightly better stabilized with modifications at the C-terminus
(PNA 15) or in the middle (PNA 14) (ΔTm= 3.0−4.4 °C).
Thus, the bulky carboxyfluorescein is tolerated only when
placed away from the PNA backbone.
To determine whether the increased affinity is at the cost of
sequence specificity, the Tm’s of modified PNAs hybridized with
DNA2 having single base mismatch in the middle of the
sequence were determined (Table 1, Figure 2). The
introduction of one base-pair mismatch significantly lowered
the Tm of the azido PNA:DNA 2 duplexes compared to that
with corresponding azido PNA:DNA 1 duplexes with cDNA.
The relative destabilization for the azm and azb-PNA:DNA 2
complexes (ΔTm= −10 to −14.6 °C) was much higher than
that for the aeg-PNA:DNA 2 duplex (ΔTm= −6 °C). These
results clearly suggest that chiral, azide-modified PNAs not only
have higher binding affinity with cDNA but also have a better
mismatch discrimination in their specificity for target cDNA.
The fluorescently labeled PNAs also show better mismatch
discrimination compared to control aeg PNA (Supporting
Information).
The effect of γ-side-chain substitution on the conformation
of PNA:DNA duplexes was examined by CD spectroscopy.
Single strand PNAs have weak or nonexistent CD signals, while
the PNA:DNA complexes exhibit characteristic CD signatures
due to the helicity induced by the chiral DNA in the hybrid.
However, the CD profiles for duplexes of azide modified PNAs
were similar to that of the unmodified PNA:DNA duplex
(Supporting Information).
THERMAL STABILITY OF PNA:DNA HYBRIDS
■
To evaluate the effect of the chiral, charge-neutral azide group
linked at Cγ-position through methylene/butylene chains on
the thermal stability of PNA:DNA duplexes, the melting
temperatures (Tm) of duplexes with complementary DNA 1
were measured by temperature-dependent UV absorbance. The
various PNAs were annealed individually with stoichiometric
amounts of antiparallel cDNA strand (DNA 1) to constitute
PNA:DNA duplexes. The sequence specific recognition of
modified PNA oligomers with cDNA was also assessed from
their binding studies with DNA 2 having a single base
mismatch. The Tm results for all PNA:DNA duplexes are shown
in Table 1.
Incorporation of neutral azide function in the Cγ-side chain
of aeg PNA backbone affects the thermal stability of the derived
PNA:DNA hybrids (Table 1, Figure 2). In the case of
azidomethylene (azm) PNAs, the PNA:DNA duplex was
enhanced by 0.2−6.3 °C depending on the site of modification.
Substitution at the C-terminus (PNA 4, ΔTm = 2.4 °C) was
found to be better than that at the N-terminus (PNA 2, ΔTm =
0.5 °C) and even better when the azm modification is in the
middle of the sequence (PNA 3, ΔTm = 6.3 °C). However,
when these two modifications were combined, the doubly
modified PNA (PNA 5) showed stability similar to that of aeg
PNA 1. In the case of longer side chain azidobutylene (azb)
PNAs, single modification at the C-terminus (PNA 8) gave
higher stability (ΔTm = 7.5 °C) than the N-terminus modified
(PNA 6) (ΔTm = 1.5 °C) similar to the shorter chain azm
modification. However, the middle modification exhibited a
stabilization (PNA 7; ΔTm = 5.8 °C) in between that of the N-
terminus (PNA 6) and C-terminus (PNA 8) modifications
unlike the case of azm, where it was the highest. The azb PNA
9 carrying double modification at the C-terminus and middle
showed significant stabilization of 7.2 °C. These results on
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Note
3-Amino-2-(Boc-amino)propanoic Acid (2a).21 A slurry of
compound N-Boc-L-asparagine (1) (5 g, 21.5 mmol), EtOAc (24 mL),
CH3CN (24 mL), H2O (12 mL), and iodobenzene diacetate (8.3 g,
25.8 mmol) was cooled and stirred at 16 °C for 30 min The
temperature was allowed to reach 20 °C, and the reaction mixture was
stirred for 4 h. It was cooled to 0 °C and filtered under vacuum. The
solid residue was washed with EtOAc and dried in vacuum to obtain
compound 2a (2.87 g, 65% yield): mp = 210−212 °C; Rf = 0.25
The relative binding affinities of azide-bearing PNAs with
cDNA was examined by their ability to invade DNA:DNA
duplex, bind to cDNA, and displace the isosequential second
DNA strand. This reaction was monitored by an ethidium
bromide (EtBr) displacement assay26 wherein the fluorescence
from the dye intercalated in the DNA duplex is decreased as
PNAs invade the duplex to form PNA:DNA duplex that cannot
bind the dye.27 The relative dye displacement efficiencies
followed by monitoring the decrease in fluorescence at 610 nm
(see the Supporting Information) for aeg (1), azb (8), and azm
(4) PNAs were 84%, 89%, and 63%, respectively, at the same
DNA duplex concentrations. These suggest that the azb-PNA
(8) which forms the most stable duplex is also the best in
displacement of the intercalated dye.
1
EtOAc/MeOH (50:50); H NMR (400 MHz, DMSO-d6) δ 7.95 (bs,
3H), 7.24−7.22 (d, J = 8 Hz, 1H), 4.24−4.19 (m, 1H), 3.22−3.17 (m,
1H), 3.02−2.98 (m, 1H), 1.39 (s, 9H); HRMS (ESI-MS) m/z calcd
for C8H16N2O4 [M + H]+ 205.1188, found 205.1198.
3-Azido-2-(Boc-amino)propanoic Acid (3a). To a stirred
solution of compound 2a (1.2 g, 5.9 mmol) in MeOH/H2O (1:1)
were added Et3N (2.5 mL, 17.9 mmol) and CuSO4·5H2O (145 mg,
0.58 mmol) in H2O (0.5 mL). To this solution was added triflyl azide
in DCM through a dropping funnel, and the reaction mixture was
stirred for 10 h. The solvent was evaporated under vacuum, and
saturated aq NaHCO3 was added to the residue and extracted with
EtOAc (2 × 20 mL). The aqueous layer was acidified to pH 2−3 by
slow addition of saturated aq KHSO4 solution and extracted with
EtOAc (3 × 30 mL). The combined EtOAc extracts were dried over
anhydrous Na2SO4, filtered, and concentrated to give compound 3a as
yellow sticky liquid and used for next step without further purification:
Rf = 0.2 EtOAc; IR (neat) 3187, 2983, 2937, 2109, 1691, 1513 cm−1.
6-Azido-2-(Boc-amino)hexanoic Acid (3b). By following a
similar procedure as above starting from N-Boc-L-lysine (2b) (246
mg, 1.0 mmol) in CH3OH/H2O (1:1), and employing Et3N (0.3 g,
0.42 mL, 3 mmol) and aq CuSO4·5H2O (12.5 mg, 0.05 mmol) (0.5
mL), triflyl azide in DCM, followed by usual workup gave compound
3b, which was used in the next step without further purification: Rf =
0.22 EtOAc; IR (neat) 3207, 2982, 2106, 1691, 1613 cm−1.
CELL PENETRATION STUDIES OF FLUORESCENTLY
LABELED PNA OLIGOMERS
■
The cell penetration abilities and the intracellular distribution
of PNAs were investigated in NIH 3T3 cell lines by confocal
microscopy employing standard cellular uptake assay, incubat-
ing the live cells with fluorescently labeled PNAs. Figure 3
Methyl 3-Azido-2-(Boc-amino)propanoate (4a). To a stirred
solution of compound 3a (1 g, 4.34 mmol) in acetone (15 mL) was
added K2CO3 (1.8 g, 13 mmol) followed by dimethyl sulfate (0.65 g;
0.5 mL, 5.2 mmol). The reaction mixture was heated to 55 °C for 4 h
under reflux condenser. Acetone was evaporated completely, and water
(30 mL) was added to the concentrate, which was then extracted with
EtOAc (3 × 40 mL). The combined organic extracts were washed with
brine, dried over anhydrous Na2SO4, filtered, and concentrated. The
residue was purified on silica gel (60−120 mesh) using petroleum
ether and EtOAc to give compound 4a as colorless liquid (0.7 g, 66%
Figure 3. Distribution of various PNAs inside NIH 3T3 cells: (A)
bright field image (B) Hoechst 33342 stained image, (C) green
fluorescent image, and (D) superimposed image of A−C.
yield): Rf = 0.55 petroleum ether/EtOAc (75:25); [α]25 +5.8 (c 1,
shows live cell images for cell penetration of PNAs azm-Tf PNA
12 and azb-Tf PNA 15 in NIH 3T3 cells, bright field image
(Figure 3A), Hoechst 33342 stained image (Figure 3B), the
green fluorescent image (Figure 3C), and the superimposed
image (Figure 3D). In comparison with control PNAs
(Supporting Information), these suggest that the cells
accumulate the modified PNAs in the cytoplasm in the vicinity
of the nucleus.
D
1
Methanol); IR (neat) 3361, 2925, 2855, 2105, 1711, 1506 cm−1; H
NMR (200 MHz, CDCl3) δ 5.38 (app d, J = 6 Hz, 1H), 4.50−4.43 (m,
1H), 3.79 (s, 3H), 3.72 (app d, J = 2 Hz, 2H), 1.45 (s, 9H); 13C NMR
(50 MHz, CDCl3) δ 170.2, 155.0, 80.4, 53.4, 52.7, 52.5, 28.1; HRMS
(ESI-MS) m/z calcd for C9H16N4O4 [M + Na]+ 267.1069, found
267.1075.
Methyl 6-Azido-2-(Boc-amino)hexanoate (4b). Using the same
procedure as above with compound 3b (4.4 g, 16.2 mmol) in acetone
(80 mL), K2CO3 (5.6 g, 40.4 mmol), and dimethyl sulfate (2.44 g;
1.88 mL, 19.4 mmol), and workup followed by column purification
over silica gel (60−120 mesh) and elution with petroleum ether and
EtOAc gave compound 4b as a yellowish liquid (3.8 g, 82% yield): Rf =
0.8 petroleum ether/EtOAc (60:40); [α]25D −16.0 (c 1, CH3OH); IR
In conclusion, it is demonstrated that the Cγ-substituted
methylene/butylene azido PNAs enable the attachment of
fluorophore through click reactions at desired sites in the PNA.
PNA analogues with a Cγ-butylene spacer chain show higher
stabilization of DNA hybrids than that from a methylene spacer
chain. The fluorescent PNA oligomers accumulate around the
nuclear membrane as seen by live cell imaging in 3T3 cells.
Since azido function does not need protection, the side-chain
conjugation strategy reported here permits multisite labeling of
same ligand in a single step to enhance the sensitivity of
detection. The outcome of the work has potential for future
utilization of azido PNAs to conjugate different ligands for
multipurpose probing various biofunctions.
1
(neat) 2925, 2856, 2095, 1707, 1512 cm−1; H NMR (200 MHz,
CDCl3) δ 5.06 (d, J = 8 Hz, 1H), 4.33−4.23 (m, 1H), 3.72 (s, 3H),
3.25 (t, J = 7 Hz, 2H), 1.76−1.53 (m, 6H), 1.42 (s, 9H); 13C NMR
(50 MHz, CDCl3) δ 173.1, 155.3, 80.0, 53.1, 52.3, 51.1, 32.2, 28.3,
28.2, 22.5; MS (MALDI-TOF) m/z calcd for C12H22N4O4 [M + K]+
325.1278, found 325.1252.
2-[(Azidomethylene)-N-Boc-amino]ethanolamine (5a). To a
stirred solution of compound 4a (3.8 g, 15.6 mmol) in absolute EtOH
(50 mL) was added NaBH4 (1.78 g, 46.8 mmol), and the reaction
mixture was stirred for 6 h under nitrogen atmosphere at room
temperature. EtOH was evaporated completely, and water (100 mL)
was added to the concentrate which was extracted with EtOAc (3 × 75
mL). The combined organic layer was washed with brine, dried over
anhydrous Na2SO4, filtered, and concentrated. The residue was
EXPERIMENTAL SECTION
See the Supporting Information for general information on the source
of materials and methods.
■
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Note
purified on silica gel (60−120 mesh) using petroleum ether and
EtOAc to give compound 5a as a colorless liquid (2.9 g, 86% yield): Rf
EtOH (20 mL) was added hydrazine hydrate (3.09 g, 3 mL, 63.6
mmol), and the reaction mixture was stirred for 4 h at room
temperature. After completion of reaction, solvent was evaporated
completely, water (30 mL) was added, and the solution extracted with
EtOAc (3 × 50 mL). The organic layer was dried over anhydrous
Na2SO4 and concentrated to give free amine (0.68 g), which was taken
in CH3CN (20 mL). To this, Et3N (0.8 g, 1.1 mL, 7.9 mmol) was
added, the mixture was stirred at 0 °C for 10 min, ethyl bromoacetate
(0.46 g, 0.31 mL, 2.86 mmol) was added dropwise, and stirring was
continued for 12 h at room temperature. Aqueous workup and
extraction with EtOAc (3 × 40 mL) and concentration gave a residue
which was purified on silica gel (100−200 mesh), eluting with
petroleum ether, and EtOAc to give compound 8a as a yellow sticky
oil (0.7 g, 73% yield): Rf = 0.79 EtOAc; [α]25D+ 8.0 (c 1, methanol);
= 0.35 petroleum ether/EtOAc (70:30); [α]25 +13.6 (c 1, CH3OH);
D
1
IR (neat) 3331, 2977, 2933, 2097, 1687, 1514 cm−1; H NMR (200
MHz, CDCl3) δ 5.06 (d, J = 6 Hz, 1H), 3.81−3.70 (m, 3H), 3.52−
3.43 (m, 2H), 2.66 (br, 1H), 1.44 (s, 9H); 13C NMR (50 MHz,
CDCl3) δ 155.7, 80.2, 62.2, 51.5, 28.3; HRMS (ESI-MS) m/z calcd for
C8H16N4O3 [M + Na]+ 239.1119, found 239.1127.
tert-Butyl (6-Azido-1-hydroxyhexan-2-yl)carbamate (5b).
Using a similar procedure as above starting from compound 4b (1.6
g, 5.6 mmol) in absolute EtOH (30 mL), NaBH4 (0.64 g, 16.8 mmol),
workup, and column chromatography purification gave compound 5b
as a colorless liquid (1.2 g, 85% yield): Rf = 0.5 petroleum ether/
EtOAc (50:50); [α]25 −12.9 (c 1, methanol); IR (neat) 3341, 2937,
D
2868, 2093, 1684, 1511 cm−1; 1H NMR (200 MHz, CDCl3) δ 4.84 (d,
J = 4 Hz, 1H), 3.56−3.47 (m, 2H), 3.49−3.47 (m, 1H), 3.24 (t, J = 6
Hz, 2H), 3.12 (br, 1H), 1.72−1.52 (m, 6H), 1.40 (s, 9H); 13C NMR
(50 MHz, CDCl3) δ 156.3, 79.5, 65.2, 52.3, 51.2, 31.0, 28.6, 28.3, 23.1;
HRMS (ESI-MS) m/z calcd for C11H22N4O3 [M + Na]+ 281.1589,
found 281.1602.
1
IR (neat) 3337, 2975, 2927, 2857, 2099, 1737, 1702, 1514 cm−1; H
NMR (400 MHz, CDCl3) δ 5.09 (br, 1H), 4.18 (q, J = 8 Hz, 2H), 3.76
(br, 1H), 3.49−3.33 (m, 4H), 2.81−2.67 (m, 2H), 2.24 (br, 1H), 1.44
(s, 9H), 1.27 (t, J = 8 Hz, 3H); 13C NMR (100 MHz, CDCl3) δ 172.2,
155.4, 79.8, 60.9, 52.5, 50.7, 50.0, 29.6, 28.4, 14.1; HRMS (ESI-MS)
m/z calcd for C12H23N5O4 [M + Na]+ 324.1647, found 324.1653.
Ethyl 2-((6-Azido-2-(Boc-amino)hexyl)amino)acetate (8b).
Using a similar procedure with compound 7b (0.8 g, 2.06 mmol) in
absolute EtOH (20 mL), hydrazine hydrate (2.06 g, 2 mL, 41.2
mmol), Et3N (0.5 g, 0.7 mL, 5 mmol), and ethyl bromoacetate (0.3 g,
0.2 mL, 1.8 mmol), aqueous workup and column chromatography
purification gave compound 8b as an oil (0.55 g, 78% yield): Rf = 0.5
3-Azido-2-(Boc-amino)propyl Methanesulfonate (6a). To an
ice-cold solution of compound 5a (2.1 g, 9.72 mmol) and Et3N (2.44
g, 3.38 mL, 24.3 mmol) in dry DCM (40 mL) was added mesyl
chloride (1.48 g, 1 mL, 12.63 mmol), and the reaction mixture was
stirred for 30 min at 0 °C under nitrogen atmosphere. To the reaction
mixture was added DCM (20 mL), which was washed with water (20
mL) and brine (20 mL). The organic layer was dried over anhydrous
Na2SO4, filtered, and concentrated to give compound 6a (2.29 g, 80%
crude yield): Rf = 0.69 petroleum ether/EtOAc (50:50) which was
used for next step without further purification.
6-Azido-2-(Boc-amino)hexyl Methanesulfonate (6b). Com-
pound 5b (3.25 g, 12.6 mmol), dry DCM (60 mL), Et3N (3.19 g, 4.4
mL, 31.5 mmol), and mesyl chloride (1.87 g, 1.27 mL, 16.38 mmol)
under similar reaction conditions, workup, and purification gave
compound 6b (3.5 g, 83% crude yield): Rf = 0.62 petroleum ether/
EtOAc (50:50) which was used for next step without further
purification.
EtOAc; [α]25 +10.6 (c 1, methanol); IR (neat) 2926, 2859, 2094,
D
1
1741, 1672, 1517 cm−1; H NMR (200 MHz, CDCl3) δ 4.74−4.71
(app d, J = 6 Hz, 1H), 4.24−4.13 (q, J = 8 Hz, 2H), 3.65−3.63 (m,
1H), 3.42−3.38 (d, J = 8 Hz, 2H), 3.30−3.24 (t, J = 6 Hz, 2H), 2.74−
2.60 (m, 2H), 1.93 (br, 1H), 1.64−1.52 (m, 6H), 1.44 (s, 9H), 1.31−
1.24 (t, J = 7 Hz, 3H); 13C NMR (50 MHz, CDCl3) δ 172.4, 155.8,
79.2, 60.8, 52.9, 51.3, 50.9, 32.8, 28.7, 28.4, 23.1, 14.2; MS (MALDI-
TOF) m/z calcd for C15H29N5O4 [M + K]+ 382.1857, found 382.1859.
Ethyl 2-(N-(3-Azido-2-(Boc-amino)propyl)-2-chloro-
acetamido)acetate (9a). To an ice-cold solution of compound 8a
(1.1 g, 3.65 mmol) in dry DCM (20 mL) and Et3N (1.1 g; 1.5 mL,
10.9 mmol) was added chloroacetyl chloride (0.45 g; 0.32 mL, 4
mmol), and the reaction mixture was stirred for 8 h. The reaction
mixture was diluted with DCM (20 mL) and washed with water (40
mL) and brine (40 mL). The dried organic layer was concentrated to
obtain a residue which was purified on silica gel (100−200 mesh)
eluting with petroleum ether and EtOAc to give compound 9a as pale
yellow sticky oil (1 g, 73% yield): Rf = 0.65 petroleum ether/EtOAc
tert-Butyl (1-Azido-3-(1,3-dioxoisoindolin-2-yl)propan-2-yl)-
carbamate (7a). To a stirred solution of compound 6a (2.25 g, 7.65
mmol) in dry DMF (30 mL) were added K2CO3 (2.11 g, 15.3 mmol)
and potassium phthalimide (5.66 g, 30.6 mmol), and the reaction
mixture was heated to 80 °C for 12 h. To this was added water (60
mL) and the solution extracted with EtOAc (3 × 50 mL). The organic
layer was washed with water (50 mL) and brine (30 mL), and the
dried organic layer on concentration gave a residue which was purified
on silica gel (100−200 mesh) using petroleum ether and EtOAc to
give compound 7a as a white solid (1.8 g, 68% yield): mp = 114−117
(50:50); [α]25 +5.2 (c 1, Methanol); IR (neat) 3340, 2977, 2928,
D
2860, 2102, 1742, 1704, 1659, 1515 cm−1; 1H NMR (400 MHz,
CDCl3) δ 5.14−5.12 (maj) 5.06−5.05 (min) (app d, J = 8 Hz, 4 Hz,
1H), 4.27−4.18 (m, 4H), 4.02 (s, 2H), 3.89−3.65 (m, 2H), 3.59−3.41
(m, 3H), 1.44 (min) 1.43 (maj) (s, 9H), 1.33−1.30 (maj) 1.29−1.27
(min) (t, J = 6 Hz, 4 Hz, 3H); 13C NMR (100 MHz, CDCl3) δ 168.8,
168.4, 167.6, 80.0, 62.2, 52.3, 51.3, 50.4, 49.1, 48.7, 40.9, 28.3, 14.1;
HRMS (ESI-MS) m/z calcd for C14H24ClN5O5 [M + Na]+ 400.1363,
found 400.1371.
°C; Rf = 0.72 petroleum ether/EtOAc (50:50); [α]25 +50.0 (c 0.5,
D
methanol); IR (neat) 3370, 2977, 2936, 2102, 1773, 1711, 1512 cm−1;
1H NMR (400 MHz, CDCl3) δ 7.88−7.86 (m, 2H), 7.74−7.72 (m,
2H), 5.04 (app d, J = 8 Hz, 1H), 4.12 (br, 1H), 3.89−3.73 (m, 2H),
3.58−3.55 (m, 2H), 1.30 (s, 9H); 13C NMR (100 MHz, CDCl3) δ
168.4, 155.3, 134.1, 131.9, 123.5, 79.9, 52.7, 49.4, 39.7, 28.1; MS
(MALDI-TOF) m/z calcd for C16H19N5O4 [M + K]+ 384.1074, found
384.1098.
Ethyl 2-(N-(6-Azido-2-(Boc-amino)hexyl)-2-chloro-
acetamido)acetate (9b). Compound 8b (1 g, 2.9 mmol) in dry
DCM (20 mL) and Et3N (0.88 g; 1.21 mL, 8.7 mmol) and
chloroacetyl chloride (0.326 g; 0.23 mL, 2.9 mmol) under a procedure
similar to that above gave compound 9b as a yellowish sticky oil (0.9 g,
74% yield): Rf = 0.67 petroleum ether/EtOAc (50:50); [α]25D −1.2 (c
1, methanol); IR (neat) 3333, 2977, 2938, 2868, 2095, 1742, 1694,
tert-Butyl (6-Azido-1-(1,3-dioxoisoindolin-2-yl)hexan-2-yl)-
carbamate (7b). Compound 6b (3.5 g, 10.4 mmol) in dry DMF
(50 mL), K2CO3 (2.87 g, 20.8 mmol) and potassium phthalimide (7.7
g, 41.6 mmol) under similar conditions as above, workup, and column
purification gave compound 7b as a white solid (3.2 g, 80% yield): mp
= 94−96 °C; Rf = 0.79 petroleum ether/EtOAc (60:40); [α]25D +20.6
(c 0.5, methanol); IR (neat) 3371, 2936, 2867, 2093, 1772, 1704,
1614, 1511 cm−1; 1H NMR (400 MHz, CDCl3) δ 7.84−7.82 (m, 2H),
7.70−7.68 (m, 2H), 4.62 (app d, J = 12 Hz, 1H), 3.97−3.93 (m, 1H),
3.70−3.60 (m, 2H), 3.27 (t, J = 8 Hz, 2H), 1.64−1.43 (m, 6H), 1.21
(s, 9H); 13C NMR (100 MHz, CDCl3) δ 168.5, 155.7, 133.9, 132.0,
123.2, 79.2, 51.2, 49.6, 42.2, 32.3, 28.5, 28.0, 23.0; MS (MALDI-TOF)
m/z calcd for C19H25N5O4 [M + K]+ 426.1544, found 426.1518.
Ethyl 2-((3-Azido-2-(Boc-amino)propyl)amino)acetate (8a).
To a stirred solution of compound 7a (1.1 g, 3.18 mmol) in absolute
1
1655, 1515 cm−1; H NMR (200 MHz, CDCl3) δ 4.76−4.72 (min)
and 4.62−4.5 (maj) (d, J = 8 Hz, 6 Hz, 1H), 4.29−4.08 (m, 4H), 4.01
(s, 2H), 3.91−3.60 (m, 2H), 3.53−3.13 (m, 3H), 1.74−1.49 (m, 6H),
1.43 (maj) and 1.42 (min) (s, 9H), 1.34−1.25 (comp, 3H); 13C NMR
(50 MHz, CDCl3) δ 168.7, 167.3, 155.5, 79.1, 61.8, 53.1, 51.0, 49.5,
48.7, 40.9, 32.0, 28.4, 28.1, 23.2, 13.9; MS (MALDI-TOF) m/z calcd
for C17H30ClN5O5 [M + K]+ 458.1573, found 458.1588.
Ethyl 2-(N-(3-Azido-2-(Boc-amino)propyl)-2-(5-methyl-2,4-
dioxo-3,4-dihydropyrimidin-1(2H)-yl)acetamido)acetate (10a).
A solution of compound 9a (1 g, 2.65 mmol) in dry DMF (15 mL)
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Note
containing K2CO3 (0.44 g, 3.18 mmol) and thymine (0.4 g, 3.18
mmol) was stirred at room temperature for 12 h. To the reaction
mixture was added water (40 mL), the solution was extracted with
EtOAc (3 × 50 mL), and the organic layer was washed with water (50
mL) and brine (30 mL). The dried organic extractupon concentration
and coulmn chromatography purification gave compound 10a as white
solid (1 g, 80% yield): Rf = 0.6 EtOAc; mp = 102−105 °C; [α]25D +9.4
(c 1, methanol); IR (neat) 3331, 2977, 2101, 1737, 1686, 1518, 1467,
1417 cm−1;1H NMR (400 MHz, CD3OD) δ 7.30−7.28 (comp, 1H),
4.79−4.50 (m, 2H), 4.4−4.1 (m, 4H), 4.1−3.8 (m, 2H), 3.75−3,55
(m, 2H), 3.45−3.35 (m, 1H), 3.31−3.24 (m, 1H), 1.88 (s, 3H), 1.45
(s, 9H), 1.34−1.31 (min) 1.29−1.27 (maj) (t, J = 6 Hz, 4 Hz, 3H);
13C NMR (100 MHz, CD3OD) δ 171.0, 170.3, 167.4, 158.2, 153.3,
mL), the free amine was coupled with carboxylic acid of incoming
monomer (A/T/G/C; 3 equiv) in DMF (500 μL) using HOBt (3
equiv), HBTU (3 equiv), and DIPEA (3 equiv). The reagents were
then removed by filtration, and the resin was washed with DMF.
Synthesis of Fluorescent PNA Oligomers on Solid Support
by Click Reaction. The resin-bound azido PNA oligomers (10 mg,
0.35 mmol/g) in DMF:pyridine (1:2) were reacted with the propyne
5(6)-carboxyfluorescein (8.66 mg, 6 equiv) in the presence of CuI (12
mg, 18 equiv), ascorbic acid (3.1 mg, 5 equiv), and DIPEA (15 μL, 24
equiv) for 24 h at room temperature. The reagents were removed by
filtration and the resin was washed with DMF and subjected to
cleavage reaction.
Purification of PNA Oligomers by Reversed-Phase HPLC.
PNA purification was carried out on Dionex ICS 3000 HPLC system.
For the purification of peptides, semipreparative BEH130 C18 (10 ×
250 mm) column was used. Purification of PNA oligomers was
performed with gradient elution method: A to 100% B in 20 min; A =
0.1% TFA in CH3CN/H2O (5:95); B = 0.1% TFA in CH3CN/H2O
(1:1) with flow rate of 3 mL/min. Control aeg, azm, and azb modified
PNA oligomers were monitored at 254 nm wavelength while all
fluorescent PNA oligomers were monitored at both 254 and 490 nm
wavelengths.
144.0, 117.2, 111.3, 81.1, 62.9, 53.2, 51.4, 51.0, 50.4, 29.1, 14.8, 12.6;
HRMS (ESI-MS) m/z calcd for C19H29N7O7 [M + Na]+ 490.2026,
found 490.2029.
Ethyl 2-(N-(6-Azido-2-(Boc-amino)hexyl)-2-(5-methyl-2,4-
dioxo-3,4-dihydropyrimidin-1(2H)-yl)acetamido)acetate (10b).
Compound 9b (1 g, 2.38 mmol) in dry DMF (15 mL), K2CO3 (0.4 g,
2.9 mmol), and thymine (0.36 g, 2.9 mmol) under a similar procedure
gave compound 10b as a white sticky solid (1 g, 85% yield): Rf = 0.63
EtOAc; [α]25 +1.7 (c 1, methanol); IR (neat) 2936, 2354, 2317,
D
2097, 1792, 1769, 1740, 1705, 1677, 1658 cm−1; 1H NMR (200 MHz,
CDCl3) δ 9.52 (maj) 9.32 (min) (br, 1H), 7.09, 7.03 (s, 1H); 5.33−
5.29 (d, J = 8 Hz, 1H), 5.02−4.71 (comp, 1H), 4.54−4.12 (m, 5H),
3.97−3.77 (comp, 1H), 3.56−3.22 (m, 5H), 1.91 (min) 1.90 (maj) (s,
3H), 1.62−1.48 (m, 6H), 1.43 (maj) 1.39 (min) (s, 9H), 1.31−1.23
(comp, 3H); 13C NMR (50 MHz, CDCl3) δ 174.8,169.1, 167.4, 164.5,
155.9, 151.2, 141.3, 141.0, 110.6, 79.5, 61.4, 52.6, 51.1, 49.3, 47.6, 31.7,
28.5, 28.3, 23.3, 14.1, 12.3; HRMS (ESI-MS) m/z calcd for
C22H35N7O7 [M + Na]+ 532.2496, found 532.2491.
UV−Tm Measurements. UV−melting experiments were carried
out on Varian Cary 300 UV spectrophotometer equipped with a
Peltier attachment. The samples for Tm measurement were prepared
by mixing the calculated amounts of respective oligonucleotides in
stoichiometric ratio (1:1, for duplex) in sodium phosphate buffer (10
mM) containing EDTA (0.1 mM) and NaCl (10 mM); pH 7.2 to
achieve a final strand concentration of 2 μM for each strand. The
samples were annealed by heating at 90 °C for 3 min followed by slow
cooling to room temperature for at least 6−8 h and then refrigerated
for at least 4 to 5 h. The samples (500 μL) were transferred to quartz
cell and equilibrated at the starting temperature for 2 min. The OD at
260 nm was recorded in steps from 20 to 85 °C with temperature
increment of 1 °C/min. Each melting experiment was repeated at least
twice. The normalized absorbance at 260 nm was plotted as a function
of the temperature. The Tm was determined from the first derivative of
normalized absorbance with respect to temperature and is accurate to
0.5 °C. The data were processed using Microcal Origin 8.5. [The
concentration of all oligonucleotides were calculated on the basis of
absorbance from the molar extinction coefficients of the corresponding
nucleobases, i.e., T = 8.8 cm2/μmol; C = 7.3 cm2/μmol; G = 11.7 cm2/
μmol, and A = 15.4 cm2/μmol.]
Circular Dichroism. CD spectra were recorded on JASCO J-815
spectropolarimeter connected with a Peltier. The calculated amounts
for PNA oligomers, and the complementary DNA1 were mixed
together in stoichiometric ratio (1:1, for duplex) in sodium phosphate
buffer (10 mM) containing EDTA (0.1 mM) and NaCl (10 mM), pH
7.2, to achieve a final strand concentration of 5 μM for each strand.
The samples were annealed by heating at 90 °C for 3 min followed by
slow cooling to room temperature for at least 6−8 h. The cooled
samples were kept at 4 °C for 4 h. The CD spectra of PNA:DNA
duplexes and single-stranded PNAs were recorded at 10 °C with an
accumulation of five scans from 300 to 190 nm using 2 mm cell, a
resolution of 0.1 nm, bandwidth of 1 nm, sensitivity of 2 m deg,
response of 2 s, and a scan speed of 50 nm/min.
2-(N-(3-Azido-2-(Boc-amino)propyl)-2-(5-methyl-2,4-dioxo-
3,4-dihydropyrimidin-1(2H)-yl)acetamido)acetic Acid (11a). To
a stirred solution of compound 10a (1 g, 2.14 mmol) in THF was
added a solution of 10% LiOH in water, and the reaction mixture was
stirred for 2 h. Solvent removal, aqueous workup with acidified
saturated KHSO4 at pH 3−4, and extraction with diethyl ether
afforded acid monomer 11a as a white solid (0.85 g, 90% yield): mp =
150−152 °C; Rf = 0.5 EtOAc/MeOH (50:50); [α]25 +11.2 (c 0.5,
D
methanol); IR (neat) 3338, 2975, 2822, 2371, 2317, 2099, 1733, 1682,
1670, 1525 cm−1; 1H NMR (400 MHz, DMSO-d6) δ 11.31 (maj) and
11.28 (min) (s, 1H), 7.27−7.26 (comp, 1H), 7.15−7.13 (maj) and
7.01−6.98 (min) (d, J = 8 Hz, 12 Hz, 1H), 4.73−4.41 (m, 2H), 4.27−
4.21 (m, 1H), 4.05−3.86 (m, 2H), 3.60−3.40 (m, 3H), 3.23−3.0 (m,
1H), 1.75 (s, 3H), 1.39 (min) and 1.38 (maj) (s, 9H); 13C NMR (100
MHz, DMSO-d6) δ 170.3, 167.4, 164.4, 155.2, 151.0, 141.9, 108.2,
78.4, 51.2, 49.4, 49.2, 48.5, 47.7, 39.5, 28.2, 11.9; HRMS (ESI-MS) m/
z calcd for C17H25N7O7 [M + Na]+ 462.1713, found 462.1710.
2-(N-(6-azido-2-(t-Boc-amino)hexyl)-2-(5-methyl-2,4-dioxo-
3,4-dihydropyrimidin-1(2H)-yl)acetamido)acetic Acid (11b). A
similar reaction with 10b (0.5 g, 0.98 mmol) in THF and 10% aq
LiOH gave the acid monomer 11b as white solid (0.4 g, 84% yield):
mp = 167−169 °C; Rf = 0.48 EtOAc/MeOH (50:50); [α]25 +1.4 (c
D
1, methanol); IR (neat) 3361, 2937, 2318, 2097, 1677, 1530 cm−1; 1H
NMR (400 MHz, DMSO-d6) δ 11.29 (maj) and 11.27 (min) (br s,
1H), 7.22 (s, 1H), 6.82 (maj) and 6.67 (min) (d, J = 8 Hz, 1H), 4.75−
4.45 (m, 2H), 4.21−3.86 (m, 3H), 3.69−3.52 (m, 2H), 3.2−2.96 (m,
2H), 1.75 (s, 3H), 1.57−1.42 (m, 6H) 1.38 (min) and 1.36 (maj) (s,
9H); 13C NMR (100 MHz, DMSO-d6) δ 170.4, 167.3, 164.4, 155.7,
151.0, 141.9, 115.7, 108.1, 78.0, 51.5, 50.9, 50.6, 49.2, 48.5, 47.7, 30.8,
28.2, 28.0, 22.9, 12.0; HRMS (ESI-MS) m/z calcd for C20H31N7O7 [M
+ Na]+ 504.2183, found 504.2179.
Ethidium Bromide Displacement Assay from dsDNA.
Calculated amounts of DNA strands were annealed to get a DNA
duplex (2 μM, 400 μL) in the above said buffer system. The duplex in
buffer was titrated with EtBr (250 μM) with incremental addition of 1
μL of solution and the fluorescence intensity at 610 nm was recorded
till it was saturated. The resulting EtBr−dsDNA complex was titrated
individually against various PNA oligomers in the aliquots of 0.4 μL
and change in fluorescence intensity at 610 nm was recorded. The
percent change observed in fluorescence intensity (I/I0) upon
displacement of EtBr was plotted against the concentration of PNA
oligomers using Microcal Origin 8.5.
Solid-Phase Synthesis Protocol. The modified and unmodified
PNA monomers were incorporated into 10-mer PNA sequence by
solid phase synthesis on L-lysine derivatized MBHA resin having 0.35
mmol/g loading value. The deprotection of N-t-Boc group from the
resin bound lysine with 50% TFA in DCM (3 × 15 min) was followed
by washing with DCM and DMF (3 × 10 mL) to give a TFA salt of
amine which was neutralized using 10% DIPEA in DCM (3 × 10 min)
to liberate free amine. After washing with DCM and DMF (3 × 10
Cellular Uptake Experiment Using Confocal Microscopy.
NIH 3T3 cells were plated in 8-well chambered coverglass in 200 μL
of Dulbecco’s Modified Eagle Medium (DMEM) containing 10% fetal
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Note
bovine serum (FBS) at the concentration of 8000 cells per well. The
cells were grown by maintaining at 37 °C in a humidified atmosphere
containing 5% CO2 for 16 h. The required amounts of fluorescent
PNA stock solutions were added to the corresponding wells to achieve
the desired final concentration of 1 μM. The cells incubated with
fluorescent PNA oligomers were maintained at 37 °C in a humidified
atmosphere containing 5% CO2 for 24 h. After the incubation period
was over, the medium was aspirated and cells were rinsed or washed
three times with 1 medium-volume equivalent of ice-cold PBS. The
cells were then replenished with 400 μL of OPTIMEM medium
containing Hoechst 33342 at a dilution of 1/1000 of the supplied
stock solution and left for 30 min at 37 °C. After incubation for 30
min, the medium containing nuclear stain Hoechst 33342 was
removed and fresh OPTIMEM medium was added to the cells. The
cells were then immediately visualized using 40× objective of Zeiss
LSM 710 laser scanning confocal microscope. The well of cells without
incubation of any PNA oligomer was also visualized as control
experiment.
(12) Ivanova, G. B.; Arzumanov, A.; Abes, R.; Yin, H.; Wood, M. J.
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ASSOCIATED CONTENT
■
S
* Supporting Information
1H and 13C NMR and mass spectra of compounds 4a−11a and
1
4b−11b; H NMR spectrum of compound 2a; IR spectra of
compounds 3a and 3b; HPLC and MALDI-TOF of PNA 1−
16; UV−melting profiles of various modified PNA:DNA
duplexes; CD and fluorescence emission spectra of various
PNA:DNA duplexes; cell uptake studies on control PNAs. This
material is available free of charge via the Internet at http://
pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Tel: 91 (20) 2590 8021. Fax: 91 (20) 2589 9790. E-mail: kn.
ganesh@iiserpune.ac.in.
■
(25) Christensen, L.; Fitzpatrick, R.; Gildea, B.; Petersen, K. H.;
Hansen, H. F.; Koch, T.; Egholm, M.; Buchardt, O.; Nielsen, P. E.;
Coull, J.; Berg, R. H. J. Peptide Sci. 1995, 3, 175−183.
(26) (a) Yeung, B. K. S.; Tse, W. C.; Boger, D. L. Bioorg. Med. Chem.
Lett. 2003, 13, 3801−3804. (b) Tse, W. C.; Boger, D. L. Acc. Chem.
Res. 2004, 37, 61−69.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
D.R.J. acknowledges an IISER Pune for award of a research
fellowship. K.N.G. is a recipient of a JC Bose Fellowship,
Department of Science and Technology, New Delhi. We thank
Dr. Mayurika Lahiri, IISER Pune, for assistance and helpful
discussions regarding cell permeability studies.
(27) Wittung, P.; Kim, S. K.; Buchardt, O.; Nielsen, P. E.; Norden, B.
Nucleic Acids Res. 1994, 22, 5371−5377.
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