GFP-like fluorophores as DNA labels for studying DNA-protein interactions

Article abstract of DOI:10.1021/jo301684b

GFP-like 3,5-difluoro-4-hydroxybenzylideneimidazolinone (FBI) and 3,5-bis (methoxy)-4-hydroxy-benzylideneimidazolinone (MBI) labels were attached to dCTP through a propargyl linker, and the resulting labeled nucleotides (dC ^MBITP and dC^FBI

Full text of DOI:10.1021/jo301684b

Article
pubs.acs.org/joc
GFP-like Fluorophores as DNA Labels for Studying DNA−Protein
Interactions
Jan Riedl,† Petra Menova,† Radek Pohl,† Petr Orsag,‡ Miroslav Fojta,‡ and Michal Hocek*,†,§
́
́
́
†Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Gilead & IOCB Research Center,
Flemingovo nam. 2, CZ-16610 Prague 6, Czech Republic
‡Institute of Biophsics, v.v.i. Academy of Sciences of the Czech Republic, Kralovopolska 135, 61265 Brno, Czech Republic
§Department of Organic Chemistry, Faculty of Science, Charles University in Prague, Hlavova 8, CZ-12843 Prague 2, Czech Republic
S
* Supporting Information
ABSTRACT: GFP-like 3,5-difluoro-4-hydroxybenzylideneimida-
zolinone (FBI) and 3,5-bis(methoxy)-4-hydroxy-benzylideneimi-
dazolinone (MBI) labels were attached to dCTP through a
propargyl linker, and the resulting labeled nucleotides (dCMBITP
and dCFBITP) were used for a facile enzymatic synthesis of
oligonucleotide or DNA probes by polymerase-catalyzed primer
extension. The MBI/FBI-labeled DNA probes exerted low
fluorescence that was increased 2−3.2 times upon binding of a
protein. The concept was demonstrated on sequence-specific
binding of p53 to dsDNA and on nonspecific binding of single
strand binding protein to an oligonucleotide. The FBI label was
also used for a time-resolved experiment monitoring a single-
nucleotide incorporation followed by primer extension by
Vent(exo-) polymerase.
bond rotation of free HBI, which was achieved by interactions
of HBI derivatives with RNA.14 Since the HBI requires
suppression of subtle motions, 3,5-difluoro-4-hydroxybenzyli-
deneimidazolinone (FBI) and 3,5-bis(methoxy)-4-hydroxy-
benzylideneimidazolinone (MBI) were found to be more
useful than HBI for inducing fluorescence.14 To the best of our
knowledge, the only example15 of a GFP-like fluorophore (2-
hydroxybenzylidene-imidazolinone linked to 2′-OH of a ribose)
was used for the labeling of oligonucleotides (ONs) showing
large Stokes shifts.
Here we report on the synthesis of MBI and FBI conjugates
with nucleoside triphosphates (dNTPs) linked via non-
conjugate propargyl tether at C-5 of cytosine and their
incorporation of DNA. We assumed that the MBI and FBI
tags might be able to increase their fluorescence upon binding
of the labeled DNA to a protein due to restricted rotation of
the fluorophores. Such mechanism would be conceptually
different from our previous solvatochromic labels.11
INTRODUCTION
■
Fluorescent labeling of biomolecules is an indispensable tool in
chemical and molecular biology.1 In nucleic acids, intrinsically
fluorescent nucleobase analogues or nucleobase-linked fluo-
rophores have been used and applied in hybridization assays,2
single-nucleotide polymorphism typing,3 monitoring of the
polarity of the microenvironment,4 and monitoring of
interactions with a ligand.5 However, very few examples of
the use of fluorescent labeling of nucleic acids for direct
detection of interactions with unlabeled proteins6,7 or peptides8
have been reported, although an analogous use in protein
labeling for the detection of peptide−protein9 or protein−
DNA10 interaction is well established. Very recently, we have
developed11 novel nucleotides bearing solvatochromic amino-
phthalimide fluorophores that were used as building blocks for
the enzymatic synthesis of labeled DNA that enhanced
fluorescence (up to 2-fold) upon binding of some proteins.
4-Hydroxybenzylideneimidazolinone (HBI) is a green-
emitting fluorophore occurring in green fluorescent protein
(GFP).12 Unlike native GFP, HBI is very weakly fluorescent in
denatured protein and as a free molecule prepared by chemical
synthesis.13 This phenomenon is caused by non-emissive
energy dissipation caused by flexibility of bond rotation of
the free HBI molecule. Relaxation to ground state by an
emissive pathway requires planarity of a molecule accompanied
by blocking of bond rotation. This condition is fulfilled in
native GFP by specific interactions within the protein. The HBI
fluorescence can also be induced by constrained blocking of
RESULTS AND DISCUSSION
■
Synthesis and Enzymatic Incorporation of MBI- and
FBI-Labeled dNTPs. Our intended strategy for the synthesis
of fluorescent ON or DNA probes was based on the
polymerase incorporation of base-modified dNTPs16 bearing
the appropriate labels. The synthesis of the modified dNTPs
Received: August 8, 2012
© XXXX American Chemical Society
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was envisaged through the direct Sonogashira cross-coupling17
of halogenated dNTPs with a corresponding fluorophore-linked
terminal acetylenes. These building blocks were prepared in
analogy14 to the synthesis of the fluorophores.
3,5-Dimethoxy- and 3,5-difluoro-4-hydroxybenzaldehydes
were condensed with N-acetylglycine and cyclized by using
acetic anhydride as a solvent to form oxazolinone derivatives
MBO (1a) and FBO (1b) in good yields (Scheme 1). The
a
Scheme 1. Synthesis of MBI- and FBI-Labeled dNTPs
Figure 1. Incorporation of dCMBITP and dCFBITP using KOD XL
DNA polymerase. PEX products contain four modifications using
temprnd16 template. Experiments are supplemented by positive control
(+) (all natural dNTPs are present) and negative control (−) (absence
of natural dCTP).
fluorophores is pH-dependent due to ionization of the phenol
group. The pKa value of MBI is 8, whereas the pKa of FBI is 5.5
due to the −I effect of fluorine atoms.14 MBI exerts a dual
emission,14 where the neutral form emits at 480 and the
deprotonated form at 540 nm. On the other hand, a
deprotonated form of FBI emits at 501 nm, while the neutral
form is almost nonfluorescent, emitting at ca. 500 nm. To test
the pH-dependence of the MBI/FBI-labeled DNA, we have
prepared ssONs bearing either four MBI or four FBI
modifications by primer extension and magnetoseparation
(vide supra). Both the UV−vis and fluorescence spectroscopy
of these modified ONs (Figures S1−S4 in Supporting
Information) showed similar features as the free fluorophores,
but the pKa values of the fluorophores linked to ON probes
were shifted to more basic due to interactions with the ON:
MBI changed its pKa from 8 to 9.3, while FBI changed the pKa
from 5.5 to 6.5 (Supporting Information). Both fluorophores
still retain the desired low intensity of fluorescence in DNA.
Study of MBI- and FBI-Labeled DNA with DNA
Binding Proteins. Similarly as in our previous study with
solvatochromic labels,11 the DNA−protein binding experiments
were conducted on p53,18 an important tumor suppressor19
and cell cycle regulator, as an example of a sequence-specific
protein binding to dsDNA and on single strand binding protein
(SSB) as an example of a non-sequence-specific protein binding
to ssONs. In addition, we tried to employ the labeled dCFBITP
for a time-resolved monitoring of their incorporation by
Vent(exo-) polymerase and of the further movement of the
enzyme along the DNA.
a
Reagents and conditions: (i) Ac2O, (ii) propargylamine, K2CO3,
EtOH (99%), (iii) Pd(OAc)2, TPPTS, CuI, EtN(i-Pr)2, water/
acetonitrile (2:1).
oxazolinones were subsequently converted to propargyl-
imidazolinones P-MBI (2a) and P-FBI (2b) by treatment
with propargylamine in moderate yields (47% and 25%,
respectively). The reaction also led to the formation of
hydrolyzed byproduct, which were removed by extraction or
by silica-gel chromatography (for 2b). The desired MBI- and
FBI-labeled dNTPs were prepared in one step by aqueous-
phase Sonogashira coupling of dCITP with P-MBI (2a) or with
P-FBI (2b) in the presence of palladium acetate, tris(3-
sulfonatophenyl)phosphine (TPPTS), CuI, and the Hunnig
base in water/acetonitrile (2:1) in moderate yields 21% and
28%, respectively (Scheme 1).
̈
Both dCMBITP and dCFBITP were tested for the
incorporation to DNA in a primer extension (PEX) experiment
using KOD XL DNA polymerase and temprnd16 as template
(Figure 1, Table 1). The DNA polymerase was able to
incorporate both dCM/FBITPs smoothly to form full length
products, double-stranded 31-bp DNA bearing four modifica-
tions. In order to isolate single-stranded ONs (ssONs), the
PEX was performed with a biotinylated template followed by
magnetoseparation22a on streptavidin-coated magnetic beads
and release under denaturating conditions.
Binding Study with p53 Protein. Interactions of DNA with
p53 protein were studied using a 50-nt oligonucleotide (pexp53
prepared from tempp53 template using dCMBITP (3a) or
dCFBITP (3b)) containing a 20-nt p53 recognition sequence
and bearing 11 dCMBI or dCFBI fluorophores (6 within and 5
outside the recognition sequence). Bovine serum albumine
(BSA), which does not bind DNA, was used as a control.
Photophysical Properties of MBI- and FBI-Labeled
DNA. Free MBI and FBI fluorophores emit very low
fluorescence, as reported earlier.14 Emission of both free
B
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a
Table 1. List of Oligo-2′-deoxyribonucleotides Used or Synthesized
oligonucleotide
oligo-2′-deoxyribonucleotide sequence
5′-CATGGGCGGCATGGG-3′
5′-CTAGCATGAGCTCAGTCCCATGCCGCCCATG-3′
5′-CATGGGCGGCATGGGACRTGAGCRTCRATGCRTAG-3′
5′-GAATTCGATATCAAG-3′
5′-GACGGTATCGATAAGAGGCATGTCTAGGCATGTCTCTTGATATCGAATTC-3′
5′-CATGGGCGGCATGGGC-3′
5′-CTAGCATGAGCTCAGGCCCATGCCGCCCATG-3′
b
primrnd
b
temprnd16
ssDNArnd16
c
b
primp53
b
tempp53
primvent
b
b
tempvent
a
In the template (temp) ONs the segments forming a duplex with the primer are underlined, and the replicated segments are in bold. For magnetic
separation of the extended primer strands, the templates were 5′-end biotinylated. Acronyms used in the text for primer extension products are
b
analogous to those introduced for the templates (e.g., ssDNA PEX product ssDNArnd16 was synthesized on temprnd16 template, etc.). Purchased
c
oligonucleotide. Model example using dCR as modification.
The resulting MBI/FBI-labeled dsDNA (3 μM) was titrated
by 0.5 or 1 equiv of p53 solution. The results revealed a
perfectly ratiometric increase of intensity of MBI and FBI
fluorescence upon binding of p53 protein (Figure 2 and Figure
S7 in Supporting Information). The increase of fluorescence
confirmed our assumption of a more hindered rotation of
fluorophore due to the interaction with the protein. On the
other hand, the addition of BSA did not increase the
fluorescence. The 2.3-fold increase of MBI fluorescence
intensity at 484 nm was also accompanied by an additional
emission shoulder with maximum around 530 nm (even higher
increase of ca. 2.6-fold at this wavelength), which originates
from the ionized form of fluorophore formed either only in the
excited state or stabilized by binding protein. The binding of
p53 to MBI-labeled DNA therefore not only increases the
emission but also causes a dual emission. On the other hand,
binding of p53 to the FBI-labeled DNA exerted a slightly lower
2.1-fold increase of fluorescence at 496 nm.
Binding Study with SSB Protein. Interactions of SSB protein
with MBI/FBI-labeled DNA were studied on a 31-nt ssON
probe bearing four MBI or FBI modifications (prepared by
PEX using pexrnd16 followed by magnetoseparation) in the
sequence. The intensity of fluorescence was monitored upon
the addition of SSB protein solution, and again BSA was used as
a control. The experiments revealed a fully ratiometric 2.0-fold
increase of fluorescence intensity of MBI/FBI-labeled ONs
upon SSB binding at 489 and 501 nm, respectively (Figure 3).
For MBI-modified ON, an additional emission band was
observed at 535 nm, originating from the ionized form of the
fluorophore. This additional emission band was even more
significant than in the case of binding of p53 protein, showing a
nearly 3.2-fold increase of fluorescence at this wavelength and
even a 3.5-fold increase at 550 nm (Figure 3). This difference
may indicate a different binding mode of the labeled ONs to
SSB protein (according to the literature,20 the ssON is coiled
around the SSB tetramer). On the other hand, MBI- or FBI-
labeled dsDNA (PEX product of pexrnd16 without magneto-
separation) exhibited very low increase of fluorescence upon
SSB binding, indicating a priority of binding of SSB protein to
ssDNA (Figures S5 and S6 in Supporting Information).
was studied using an equimolar amount of Vent(exo-) DNA
polymerase (Figure 4). The fluorescence spectra were
measured immediately and then every minute after the addition
of the dCFBITP until the change of fluorescence was completed.
In the second stage, a mixture of natural dNTPs was added,
which enabled the enzyme to continue the PEX and finish the
DNA synthesis. Again, the fluorescence spectra were measured
every minute until the change had finished. Figure 4d shows the
expected increase of fluorescence upon the incorporation of the
dCFBITP when the polymerase remains bound to the active site
and thus interacts with the FBI label. The reaction and
equilibrium of polymerase binding takes ca. 8 min to reach the
maximum fluorescence enhancement (ca. 2.1-fold). Then, after
the addition of the natural dNTPs (Figure 4e), the polymerase
continued the synthesis of the DNA and moved along the
duplex, releasing the FBI fluorophore. This caused a rapid
decrease of fluorescence which reached the original low level
within ca. 2 min. This experiment enables a direct monitoring
of an enzymatic incorporation of a nucleotide and the following
primer extension using an unmodified enzyme (this arrange-
ment is much easier than the FRET techniques requiring
labeling of both components21).
CONCLUSIONS
■
GFP-like hydroxybenzylideneimidazolinone labels MBI and
FBI were studied as potential DNA labels for the studying of
interactions with proteins. MBI- and FBI-labeled dCTPs
(dCMBITP, dCFBITP) were prepared by aqueous Sonogashira
coupling of propargyl-MBI or -FBI with halogenated dCITP in
moderate yields. Both modified dCM/FBITPs were good
substrates for Vent(exo-) and KOD XL polymerases and
were incorporated to DNA by primer extension, enabling a
simple synthesis of ssONs or dsDNA probes containing several
site-specific MBI or FBI labels attached at the C-5 position of
cytosine(s). Both fluorophores and the labeled DNA showed
low fluorescence, which increased by 2.0−3.5 times upon
binding of a protein to the ON or DNA probe due to hindered
rotation of the fluorophores. The concept was proved both on a
p53 to a dsDNA containing a specific recognition sequence and
on a non-sequence-specific binding of SSB to ssON. The FBI
label exerted simple increase of fluorescence at the same
wavelength, whereas the MBI label showed a dual fluorescence
with an additional emission band (at 530 or 535 nm) due to
phenol deprotonation. Studying and monitoring of other DNA-
binding proteins will continue in our laboratory.
Monitoring of Incorporation of a dNTP and Primer
Extension by Vent(exo-) DNA Polymerase. The last goal was
to use the GFP-like labels for a time-resolved study of an
enzymatic reaction, i.e., incorporation of a modified dNTP and
movement of the polymerase along the DNA. For this purpose,
only the FBI label was selected due to its simple emission
behavior. The experiment was performed in two stages. At first,
a single incorporation of the labeled dCFBITP into the primer
FBI fluorophore was also successfully applied for a time-
resolved detection of a single nucleotide incorporation by DNA
polymerase (increase of fluorescence) and for the observation
C
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Figure 3. (a) Scheme of PEX incorporation of dCMBITP and dCFBITP
to ssON and binding of SSB. Photophysical properties of (b) dCMBI
or (c) dCFBI-labeled ON upon binding of SSB protein.
-
Figure 2. (a) Scheme of PEX incorporation of dCMBITP and dCFBITP
to dsDNA and binding of p53. Photophysical properties of (b) dCMBI
or (c) dCFBI-labeled DNA upon binding of p53 protein.
-
sample solutions in D2O, DMSO-d6, or CDCl3. Chemical shifts (in
ppm, δ scale) were referenced as follows: CDCl3 solutions, 1H
referenced to TMS (δ = 0 ppm), 13C referenced to the solvent signal
1
(δ = 77.0 ppm); DMSO-d6 solutions, H referenced to the residual
solvent signal (δ = 2.50 ppm), 13C referenced to the solvent signal (δ =
39.7 ppm); D2O solutions, referenced to dioxane as an internal
standard (δ(1H) = 3.75 ppm, δ(13C) = 69.3 ppm). 31P NMR spectra
were referenced to the phosphate buffer signal (δ = 2.35 ppm). 19F
NMR spectra were referenced to C6F6 as an external standard (δ =
−163.0 ppm). Coupling constants (J) are given in Hz. NMR spectra of
dNTPs were measured in phosphate buffer at pH 7.1. Complete
assignment of all NMR signals was achieved by using a combination of
H,H-COSY, H,C-HSQC, and H,C-HMBC experiments. Semiprepar-
ative separation of nucleoside triphosphates was performed by HPLC
on a column packed with 10 μm C18 reversed phase (Phenomenex,
Luna C18 (2)). High resolution mass spectra were measured using ESI
ionization technique and Orbitrap analyzer. Mass spectra of function-
of a further movement of the enzyme during primer extension
(decrease of fluorescence). This approach using just a single
modified dNTP is much simpler and more straightforward than
standard FRET techniques for the studying of the primer
extension. The concept certainly has a potential for studying of
other enzymatic reactions, e.g., DNA repair of methylation.
Studies along these lines will continue in our laboratories.
EXPERIMENTAL SECTION
■
NMR spectra were recorded on a 600 MHz (600.1 MHz for 1H, 150.9
MHz for 13C) or a 500 MHz (499.8 or 500.0 MHz for 1H, 470.3 MHz
for 19F, 200.3 MHz for 31P, 125.7 MHz for 13C) spectrometer from
D
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Figure 4. Scheme of time-resolved monitoring of primer extension. (a) Primer, template, dCFBITP, and Vent(exo-) are mixed, (b) single-nucleotide
incorporation occurs, and (c) after the addition of a mixture of natural dNTPs, the PEX continues. Time-resolved fluorescence spectroscopy of (d)
single-nucleotide incorporation dCFBI to DNA, followed by (e) PEX after the addition of natural dNTPs.
6
alized DNA were measured using MALDI-TOF ionization with
nitrogen laser.
MHz, CD3OD): 2.50 (d, 3H, J = 0.6, CH3-2 exchageable); 2.81 (t,
4
4
1H, J = 2.5, HC); 4.46 (d, 2H, J = 2.5, CH2N); 6.92 (s, 1H,
HC); 7.79 (m, 2H, H-o-C6H2F2OH). 13C NMR (125.7 MHz,
CD3OD): 15.1 (CH3-2); 30.1 (CH2N); 73.9 (HCC−); 78.3 (-C
CH); 116.4, 116.6 (2 × t, JC,F = 6.2, CH-o-C6H2F2OH); 125.9 (t, JC,F
= 9.3, C-i-C6H2F2OH); 126.8 (CH); 138.0 (t, JC,F = 16.6, C-p-
C6H2F2OH); 138.8 (C-4-imid); 153.5 (dd, JC,F = 242.2, 7.2, C-m-
C6H2F2OH); 163.8 (C-2-imid); 170.7 (C-5-imid). 19F{1H} NMR
(470.3 MHz, CD3OD): −131.79. MS (ESI+): m/z (%) 277 (50) [M +
H]+, 299 (100) [M + Na]+; HR-MS (ESI+) for C14H11O2N2F2: [M +
H]+ calculated 277.07831, found 277.07822.
General Procedure for the Synthesis of N-Propargyl-4-
hydroxy-3,5-dimethoxybenzylidene Imidazolinones. Benzyli-
dene oxazolinones14 1a or 1b were refluxed with 99% ethanol (7.5
or 15 mL), propargyl amine (1.5 equiv), and potassium carbonate (1.5
equiv) for 3 h. After cooling, the formed orange precipitate was filtered
and washed by cold ethanol. The precipitate was redissolved in a 1:1
mixture of ethyl acetate and 500 mM sodium acetate (pH = 3.0). The
organic layer was separated, and the solvent was removed under
reduced pressure.
(Z)-4-(3,5-Dimethoxy-4-hydroxybenzylidene)-2-methyl-1-
propargyl-imidazoline-5-one (P-MBI, 2a). P-MBI (2a) was
prepared according to the general procedure from 1a (1.12 g, 4.2
mmol, 1 equiv), propargylamine (0.34 g, 6.3 mmol, 1.5 equiv), and
potassium carbonate (0.86 g, 6.3 mmol, 1.5 equiv). The product was
isolated as an orange solid (601 mg, 47%). Mp 180−185 °C. 1H NMR
(499.8 MHz, DMSO-d6): 2.42 (d, 3H, 6J = 0.6, CH3-2); 3.36 (t, 1H, 4J
General Procedure for Sonogashira Cross-Coupling of Base-
Halogenated Nucleoside Triphosphates Analogues (dNITPs)
with Propargyl MBI and FBI. A mixture of H2O/CH3CN (2:1, 2
mL) was added to an argon-purged flask containing dCITP (0.05
mmol), an acetylene 2a or 2b (0.075 mmol, 1.5 equiv), and CuI (0.95
mg, 0.005 mmol, 10 mol %). In a separate flask, Pd(OAc)2 (0.56 mg,
0.0025 mmol, 5 mol %) and P(Ph-SO3Na)3 (3.59 mg, 0.00625 mmol,
2.5 equiv to Pd) were combined, evacuated, and purged with argon,
followed by the addition of H2O/CH3CN (2:1, 0.5 mL). The mixture
of the catalyst was then injected to the reaction mixture, N,N-
diisopropylethylamine (43 μL, 0.25 mmol, 5 equiv) was added, and
reaction mixture was stirred at 55 °C for 45 min. After cooling to room
temperature, 200 μL of 0.5 M EDTA solution was added to the
reaction mixture to remove the palladium catalyst. The products were
isolated by C18 reverse phase column chromatogryphy using water−
methanol (5−100%) containing 0.1 M TEAB buffer as eluent. The
dNTPs were converted to sodium salt using ionex Dowex-50,
evaporated and lyophilized.
4
= 2.5, HCC); 3.80 (s, 6H, CH3O); 4.45 (d, 2H, J = 2.5, CH2N);
6.97 (s, 1H, CH=); 7.64 (s, 2H, H-o-C6H2OH(OMe)2). 13C NMR
(125.7 MHz, DMSO-d6): 15.7 (CH3-2); 29.14 (CH2N); 56.2
(CH3O); 74.8 (HCC); 78.7 (CCH); 110.5 (CH-o-C6H2OH-
(OMe)2); 124.5 (C-i-C6H2OH(OMe)2); 127.5 (CH); 135.8 (C-4-
imid); 139.1 (C-p-C6H2OH(OMe)2); 148.0 (C-m-C6H2OH(OMe)2);
160.8 (C-2-imid); 168.8 (C-5-imid). MS (ESI+): m/z (%) 301 (100)
[M + H]+, 323 (10) [M + Na]+; HR-MS (ESI+) for C16H16O4N2Na:
[M + Na]+ calculated 323.10023, found 323.10025.
(Z)-4-(3′,5′-Difluoro-4′-hydroxybenzylidene)-2-methyl-1-
propargylimidazoline-5-one (P-FBI, 2b). P-FBI (2b) was prepared
according to the general procedure from 1b (500 mg, 2.09 mmol, 1
equiv), propargyl amine (0.17 g, 3.1 mmol, 1.5 equiv), and potassium
carbonate (0.43 g, 3.1 mmol, 1.5 equiv). The separation required
additional purification, using silica gel column chromatography
(hexane/ethyl acetate 0−20% as eluent). The product was isolated
5-{3′′′′-[(Z)-4′′-(3′′′,5′′′-Dimethoxy-4′′′-hydroxybenzyli-
dene)-2′′-methylimidazoline-5′′-one-1′′-N-yl]-1′′-propyn-1′′′′-
yl}-2′-deoxycytidine-5′-O-triphosphate (dCMBITP, 3a). dCMBITP
was prepared according to the general procedure, using dCITP (33
mg, 0.05 mmol, 1 equiv) and 2a (22.5 mg, 0.075 mmol, 1.5 equiv).
1
1
as yellow solid (144 mg, 25%). Mp 204−207 °C. H NMR (499.8
The product was isolated as yellow solid (13.4 mg, 21%). H NMR
E
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(499.8 MHz, D2O, pD = 7.1, phosphate buffer): 2.28 (dt, 1H, Jgem
=
CTAGCATGAGCTCAGGCCCATGCCGCCCATG-3′, 100 μM, 2.4
μL), primer (primvent 5′-CATGGGCGGCATGGGC-3′, 100 μM, 2.4
μL), dCFBITP (40 μM, 7.8 μL), ThermoPol reaction buffer (10×, 12
μL) and water (20.4 μL) were mixed. Next, the preconcentrated
solution of Vent(exo-) was added, and the fluorescence was measured
immediately and then after every minute until the fluorescence
stopped increasing (after 8 min). Then a mixture of natural dNTPs (4
mM, 6 μL) was added to the reaction mixture, and the fluorescence
was measured every minute until the change in fluorescence intensity
was completed (2 min).
Primer Extension. Primer Extension for Analysis by
Polyacrylamide Gel Electrophoresis. The reaction mixture (20
μL) contained primer (0.15 μM), template (0.22 μM), natural dNTPs
(200 μM), dNRTPs, buffer, and DNA polymerase (0.1 U). The
reaction mixture was incubated for 15 min (30 min for quadruple-
modified ONs) at 60 °C and analyzed by polyacrylamide gel
electrophoresis. The primers were 32P-prelabeled at 5′-end to allow
radiographic detection.
Preparative Primer Extension for Fluorescence Studies.
Reaction mixture (500 μL) for the oligonucleotide preparation
contained primer (6.6 μM), 5′-biotinylated template (6.6 μM),
dNTPs (200 μM), dNRTPs (200 μM), 10x buffer, and KOD XL
DNA polymerase (7 U). The reaction was incubated for 40 min.
Biotinylated templates were used to allow magnetoseparation.
Isolation and Characterization of Single-Strand Oligonucle-
tides by Magnetoseparatic Procedure. MagPrep P-25 (50 μL,
Streptavidin Particles stock solution from Novagen) was washed three
times with 450 μL of buffer (0.3 M NaCl, 10 mM TRIS, pH = 7.4).
The reaction mixture containing 0.3 M NaCl was added to a
suspension of magnetic beads. The suspension was shaken for 40 min
at room temperature, allowing binding of oligonucleotides to MagPrep
beads. The beads were washed three times with 500 μL of PBS
solution (0.14 M NaCl, 3 mM KCl, 4 mM sodium phosphate pH =
7.4), three times with 500 μL of TRIS buffer (0.3 M NaCl, 10 mM
TRIS, pH = 7.4), and three times with 500 μL of deionized water.
Single-stranded oligonucleotides were released by shaking and heating
the sample to 60 °C for 2 min, followed by shaking with additional
magnetic beads in Tris buffer to remove small amounts of biotinylated
template released during heating at 60 °C in deionized water. Each
medium exchange was performed using a magnetoseparator (Dynal,
Norway). The oligonucleotides were desalted and concentrated using
Amicon Ultra 0.5 Centrifugal Filter Unit (cutoff 3 kDa). Character-
ization of oligonucleotides by MS (MALDI-TOF): [M + H]+
pexrnd16(dCMBI) calcd 10810.5, found 10810.2; pexrnd16(dCFBI) calcd
10715.1, found 10716.6.
14.0, J2′b,1′ = J2′b,3′ = 6.7, H-2′b); 2.42 (ddd, 1H, Jgem = 14.0, J2′a,1′ = 6.3,
J2′a,3′ = 4.0, H-2′a); 2.51 (m, residual signal of exchengable CH3-2);
3.89 (s, 6H, CH3O); 4.20 (m, 3H, H-4′,5′); 4.58 (dt, 1H, J3′,2′ = 6.7,
4.0, J3′,4′ = 4.0, H-3′); 4.70 (s, 2H, CH2N); 6.17 (dd, 1H, J1′,2′ = 6.7,
6.3, H-1′); 6.86 (s, 1H, CH); 7.28 (s, 2H, H-o-C6H2OH(OMe)2);
8.13 (s, 1H, H-6). 13C NMR (125.7 MHz, D2O, pD = 7.1, phosphate
buffer): 17.1 (CH3-2); 33.6 (CH2N); 41.9 (CH2-2′); 58.8 (CH3O);
68.0 (d, JC,P = 5.9, CH2-5′); 73.1 (CH-3′); 77.2 (pyrimid-CC-imid);
88.2 (d, JC,P = 9.0, CH-4′); 89.2 (CH-1′); 91.8 (pyrimid-CC-imid);
94.2 (C-5); 112.7 (CH-o-C6H2OH(OMe)2); 127.5 (C-i-C6H2OH-
(OMe)2); 133.1 (CH); 137.2 (C-4-imid); 140.7 (C-p-C6H2OH-
(OMe)2); 148.1 (CH-6); 150.2 (C-m-C6H2OH(OMe)2); 158.6 (C-2);
165.1 (C-2-imid); 167.7 (C-4); 173.4 (C-5-imid). 31P{1H} NMR
(202.3 MHz, D2O, pD = 7.1, phosphate buffer, ref(phosphate buffer)
= 2.35 ppm): −21.34 (t, J = 20.0, Pβ); −9.99 (d, J = 20.0, Pα); −7.74
(d, J = 20.0, Pγ). MS (ESI−): m/z (%) 684.2 (30) [M + 2H −
PO3H]−, 764.2 (100) [M + 2H]−, 786.2 (30) [M + H + Na]−; HR-
MS (ESI−) for C25H29O17N5P3: [M + 2H]− calculated 764.07768,
found 764.07615.
5-{3′′′′-[(Z)-4′′-(3′′′,5′′′-Difluoro-4′′′-hydroxybenzylidene)-
2′′-methylimidazoline-5′′-one-1′′-N-yl]-1′′′′-propyn-1′′′′-yl}-
2′-deoxycytidine-5′-O-triphosphate (dCFBITP, 3b). dCFBITP was
prepared according to the general procedure, using dCITP (33 mg,
0.05 mmol, 1 equiv) and 2b (20.7 mg, 0.075 mmol, 1.5 equiv). The
product was isolated as yellow solid (11.5 mg, 28%). 1H NMR (499.8
MHz, D2O, pD = 7.1, phosphate buffer): 2.30 (dt, 1H, Jgem = 14.0,
J
2′b,1′ = J2′b,3′ = 6.4, H-2′b); 2.43 (ddd, 1H, Jgem = 14.0, J2′a,1′ = 6.4, J2′a,3′
= 4.1, H-2′a); 2.50 (m, residual signal of exchengable CH3-2); 4.20 (m,
3H, H-4′,5′); 4.59 (dt, 1H, J3′,2′ = 6.4, 4.1, J3′,4′ = 4.1, H-3′); 4.72 (s,
2H, CH2N); 6.20 (t, 1H, J1′,2′ = 6.4, H-1′); 7.04 (s, 1H, CH); 7.57
(m, 2H, H-o-C6H2F2OH); 8.14 (s, 1H, H-6). 13C NMR (125.7 MHz,
D2O, pD = 7.1, phosphate buffer): 17.3 (CH3-2); 33.6 (CH2N); 41.8
(CH2-2′); 67.9 (d, JC,P = 5.5, CH2-5′); 73.0 (CH-3′); 77.1 (pyrimid-
CC-imid); 88.2 (d, JC,P = 8.8, CH-4′); 89.2 (CH-1′); 92.0 (pyrimid-
CC-imid); 94.3 (C-5); 118.41, 118.6 (2 × t, JC,F = 6.6, CH-o-
C6H2F2OH); 119.0 (t, JC,F = 9.8, C-i-C6H2F2OH); 133.7 (CH);
135.5 (C-4-imid); 148.1 (CH-6); 151.8 (br, C-p-C6H2F2OH); 157.9
(dd, JC,F = 237.9, 10.7, C-m-C6H2F2OH); 158.7 (C-2); 163.6 (C-2-
imid); 167.75 (C-4); 173.5 (C-5-imid). 31P{1H} NMR (202.3 MHz,
D2O, pD = 7.1, phosphate buffer, ref(phosphate buffer) = 2.35 ppm):
−21.30 (t, J = 19.6, Pβ); −10.26 (d, J = 19.6, Pα); −6.88 (d, J = 19.6,
Pγ). 19F{1H} NMR (470.3 MHz, D2O, pD = 7.1, phosphate buffer):
−131.70. MS (ESI−): m/z (%) 581.2 (20) [M + 2H-2PO3H]−, 603.2
(40) [M + H + Na − 2PO3H]− 660.2 (8) [M + 2H − PO3H]−, 740.1
(3) [M + 2H]−; HR-MS (ESI−) for C23H23O15N5F2P3: [M + 2H]−
calculated 740.03770, found 740.03726.
ASSOCIATED CONTENT
* Supporting Information
■
Binding Study Using SSB Protein. SSB Protein from E. coli was
purchased from Sigma-Aldrich and concentrated 6x using Amicon
Ultra 0.5 Centrifugal Filter Unit (cutoff 3KDa). The measurements
were performed in 20 mM phosphate buffer pH 7.4 using three
parallel samples for exact comparison (DNA mixed with binding
protein, DNA diluted with buffer, and control sample − DNA mixed
with BSA and glycerol). An appropriate amount of glycerol was added
to negative control to refine the data from the effect of glycerol present
in the stock solution of SSB protein. BSA was used as control protein.
The measurements were performed with an AMINCO Bowman series
2 spectrofluorometer.
Binding Study Using p53 Protein. Wild type human full length
p53 proteins were expressed in E. coli BL21/DE3 and purified as
described previously.22 The measurements were performed in 50 mM
KCl, 5 mM Tris pH 7.6, 2 mM DTT, 0.01% Triton-X100, using three
parallel samples for exact comparison (DNA mixed with binding
protein, DNA diluted with buffer, and control sample − DNA mixed
with BSA). BSA was used as control protein. The measurements were
performed using a PC-1 steady-state ISS spectrofluorometer.
S
Additional figures of fluorescence and UV−vis spectra and
copies of all NMR spectra and MALDI-TOF spectra. This
material is available free of charge via the Internet at http://
pubs.acs.org/.
AUTHOR INFORMATION
Corresponding Author
*E-mail: hocek@uochb.cas.cz.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by the Academy of Sciences of the
Czech Republic (RVO: 61388963 and 68081707), by the
Czech Science Foundation (203/09/0317 and P206/12/
G151), and by Gilead Sciences, Inc. (Foster City, CA, U.S.A.).
Binding Study Using Vent(exo-) DNA Polymerase. Vent(exo-)
(2 U/μL, 0.5 μM, 500 μL) was diluted with water (200 μL) and
concentrated on an Amicon Ultra Centrifugal Filter Unit (cutoff 3
kDa, Millipore) to the final volume of 75 μL. Template (tempvent 5′-
DEDICATION
Dedicated to the memory of Dr. Detlef Schroder.
■
̈
F
dx.doi.org/10.1021/jo301684b | J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
Article
(g) Baccaro, A.; Steck, A.-L.; Marx, A. Angew. Chem., Int. Ed. 2012, 51,
254−257.
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dx.doi.org/10.1021/jo301684b | J. Org. Chem. XXXX, XXX, XXX−XXX