Design, synthesis, and polymerase-catalyzed incorporation of click-modified boronic acid-TTP analogues

Article abstract of DOI:10.1002/asia.201100229

DNA molecules are known to be important materials in sensing, aptamer selection, nanocomputing, and construction of unique architectures. The incorporation of modified nucleobases affords unique DNA properties for applications in areas that would otherwise be difficult or not possible. Earlier, we demonstrated that the boronic acid moiety can be introduced into DNA through polymerase-catalyzed reactions. In order to study whether such incorporation by polymerase is a general phenomenon, we designed and synthesized four boronic acid-modified thymidine triphosphate (TTP) analogues. The synthesis of certain analogues was through the use of a single dialkyne tether for both the Sonogashira coupling with thymidine and the later Cu-mediated [3+2] cycloaddition for linking the boronic acid moiety. This approach is much more efficient than the previously described method, and paves the way for the preparation of a large number of boronic acid-modified TTPs with a diverse set of structural features. All analogues showed very good stability under polymerase chain reaction (PCR) conditions and were recognized as a substrate by DNA polymerase, and thus incorporated into DNA.

Full text of DOI:10.1002/asia.201100229

DOI: 10.1002/asia.201100229
Design, Synthesis, and Polymerase-Catalyzed Incorporation of
Click-Modified Boronic Acid–TTP Analogues
Yunfeng Cheng, Chaofeng Dai, Hanjing Peng, Shilong Zheng, Shan Jin, and
Binghe Wang*[a]
On the occasion of the 10th anniversary of click chemistry
Abstract: DNA molecules are known
to be important materials in sensing,
aptamer selection, nanocomputing, and
construction of unique architectures.
The incorporation of modified nucleo-
bases affords unique DNA properties
for applications in areas that would
otherwise be difficult or not possible.
Earlier, we demonstrated that the bor-
onic acid moiety can be introduced
into DNA through polymerase-cata-
lyzed reactions. In order to study
whether such incorporation by poly-
merase is a general phenomenon, we
designed and synthesized four boronic
acid-modified thymidine triphosphate
(TTP) analogues. The synthesis of cer-
tain analogues was through the use of a
single dialkyne tether for both the So-
nogashira coupling with thymidine and
the later Cu-mediated [3+2] cycloaddi-
tion for linking the boronic acid
moiety. This approach is much more ef-
ficient than the previously described
method, and paves the way for the
preparation of a large number of bor-
onic acid-modified TTPs with a diverse
set of structural features. All analogues
showed very good stability under poly-
merase chain reaction (PCR) condi-
tions and were recognized as a sub-
strate by DNA polymerase, and thus
incorporated into DNA.
Keywords: Boronic acid
·
click
chemistry · DNA · glycoproteins ·
polymerase chain reaction
Introduction
ty with diols, single hydroxy groups, as well as other nucleo-
philes/Lewis bases.[7] One of our long-standing interests is
developing an aptamer selection platform specifically for
biologically important carbohydrates and glycoproteins
through the incorporation of a boronic acid group into
DNA.[7f,8] This is based on the central hypothesis that the in-
corporation of a boronic acid-modified nucleotide into
DNA would allow for enhanced recognition of carbohydrate
moieties, which contain many hydroxyl groups.[9] Along this
line, we have previously reported the design and synthesis
of a thymidine analogue (B-TTP (1), Figure 1) modified
with 8-quinolinylboronic acid at the 5-position, which can be
introduced into DNA through polymerase-catalyzed reac-
tions, as well as the feasibility of boronic acid-modified
DNA aptamer selection for biological important glycopro-
teins.[8–9] In addition, there are boronic acids that change flu-
orescent properties upon binding.[7f] Incorporation of such
boronic acids allows DNA to be used in sensing applications
without the need for an additional reporting unit.[8b] As a
specific example, we have also demonstrated the synthesis
and incorporation of a long wavelength boronic acid-modi-
fied TTP (NB-TTP, Figure 1), which shows fluorescence in-
tensity change upon carbohydrate addition. In order to ex-
The same properties that afford DNA the kind of unique
features suitable as genomic materials also allow it to be
used in a wide variety of applications such as nanosensing,[1]
aptamer selection,[2] nanocomputing,[3] and reaction encod-
ing.[4] Along these lines, modifications of nucleobases often
endow DNA with additional properties for enhanced appli-
cations.[5] For example, 5-position modified thymidine ana-
logues have been widely used in aptamer selections.[6] On
the other hand, it is also well-known that boronic acid is one
of the most commonly used building blocks for the design of
chemosensors for carbohydrates, owing to its intrinsic affini-
[a] Y. Cheng, Dr. C. Dai, H. Peng, Dr. S. Zheng, Dr. S. Jin,
Prof. B. Wang
Department of Chemistry and Center for
Diagnostics and Therapeutics
Georgia State University
Atlanta, GA 30303 (USA)
Fax : (+1)404-413-5543
E-mail: Wang@gsu.edu
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/asia.201100229.
Chem. Asian J. 2011, 6, 2747 – 2752
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FULL PAPERS
Figure 1. Chemical structures of B-TTP (1) and NB-TTP.
Figure 2. Structures of B-TTP analogues 2–5.
amine the generality of boronic acid incorporation and to
broaden the application of boronic acid-modified DNA, we
are interested in studying the incorporation of additional
boronic acids with different structural features. At the same
time, we are also interested in developing a synthetic ap-
proach which is more efficient than the previously described
method and would allow for easy analogue synthesis.[7f] The
resulting increase in structural diversity of boronic acid-
modified thymidine analogues will be very important in
future applications such as aptamer selection for biologically
important carbohydrate biomarkers as well as glycoproteins.
ic acid (7 or 8, Scheme 1) were easily obtained from their
bromomethylphenylboronic acid precursors (9 or 10,
Scheme 1), respectively. Then coupling of the appropriately
substituted azidomethylphenylboronic acid (7 or 8) through
a
copper(I)-catalyzed
azide–alkyne
cycloaddition
(CuAAC),[10] gave 2 and 3 in 31% yield after HPLC purifi-
cation.
The key design in linking the boronic acid moiety to the
5-position of thymidine is the availability of a terminal
alkyne group after the initial coupling. In order to shorten
the synthesis, we designed an approach by using a single dia-
lkyne tether for both the Sonogashira coupling with thymi-
Results and Discussion
Design and Synthesis
In our initial design, we were
interested in the introduction of
ortho- and meta-substituted
phenylboronic acid, which are
much easier to synthesize than
the previously used boronic
acids (B-TTP (1) and NB-TTP,
Figure 1). In applications where
the fluorescent properties are
not important, these phenylbor-
onic acid-modified thymidines
would be very useful and easy
to prepare. Figure 2 shows the
structures of the new analogues.
In the preparation of these new
analogues, we initially followed
the general procedures devel-
oped for the synthesis of B-
TTP (1) (Figure 1). Thus, M-
TTP (Scheme 1) was synthe-
sized following a four-step pro-
cedure starting from 5-iodo-2’-
deoxyuridine (6, Scheme 1) as
published previously.[8a] Substi-
tuted azidomethylphenylboron- Scheme 1. Synthesis of B-TTP analogues 2 to 5.
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Incorporation of Click-Modified Boronic Acid–TTP Analogues
dine and the later CuAAC for linking the boronic acid
moiety. Such a design shortens the synthesis by two steps
and simplifies the structural features of the side chain. Spe-
cifically, M(C)-TTP (Scheme 1) was prepared through Sono-
gashira coupling of 5-iodo-2’-deoxyuridine (6, Scheme 1)
with 1,7-octadiyne, and the subsequent triphosphorylation
was achieved using a modified procedure.[5a,b] Then, CuAAC
led to the final products, 4 and 5, in 29% and 14% yield, re-
spectively, after HPLC purification.
Stability Test of B-TTP Analogues under PCR Conditions
After obtaining the B-TTP analogues, we first studied
whether these analogues were stable under polymerase
chain reaction (PCR) conditions needed for polymerase-
mediated incorporation. HPLC was used to monitor the sta-
bility of the synthesized B-TTP analogues 2–5 (Figure 2).
The results showed that all B-TTP analogues are very stable
under PCR conditions including three rounds of thermal cy-
cling at 948C for 2 min, then 948C for 1 min, 468C for
1 min, and 728C for 1 min, and denaturing treatment (958C
for 7 min). The HPLC chromatograms of B-TTP analogues
2–5 are shown in Figures S1–S4 (Supporting Information).
No noticeable degradation was observed.
Figure 3. Incorporation of B-TTP analogues by Klenow fragment:
1) Primer only; 2) Primer +dNTPs, no Klenow fragment; 3) Primer+Kle-
now fragment, no dNTPs; 4) Primer+Klenow fragment+dNTPs; 5) using
B-TTP analogue 2 instead of dTTP in 4); 6) using B-TTP analogue 3 in-
stead of dTTP in 4); 7) using B-TTP analogue 4 instead of dTTP in 4);
8) using B-TTP analogue 5 instead of dTTP in 4).
To further confirm the results, besides PAGE analysis, we
also conducted matrix-assisted laser desorption/ionization
(MALDI) analyses of the primer extension product. B-TTP
analogue 2 was chosen as an example. From the MALDI
spectra (Figures S5–S6, Supporting Information), the follow-
ing results were obtained. In the control reaction, full exten-
sion of the primer using natural dNTPs yielded a DNA with
m/z of 6519 (calculated [M+H]+: 6519) as the extended
strand peak in the MALDI analysis. On the other hand,
when B-TTP analogue 2 was used instead of TTP, primer
extension yielded a DNA with m/z of 6772 and m/z of 6787
as new peaks for the corresponding extended strand. Each
was assigned as the deborylated (calc. [M+HÀHBO2]+:
6771) and oxidative deborylated sequence (calc.
[M+HÀHBO]+: 6787). Such behavior in MALDI analysis is
common in our past experience with boronic acid com-
pounds.[8] Results obtained further confirmed the full incor-
poration of the boronic acid-modified TTP analogues.
Incorporation of B-TTP Analogues by Klenow Fragment-
Catalyzed Primer Extension
Next, we studied whether the B-TTP analogues 2–5 could
be incorporated into DNA in an enzyme-catalyzed reaction,
as we have done previously with 1 (Figure 1). Specifically,
primer extension, using 2–5 and the Klenow fragment, was
conducted using a short sequence of 21-nucleotides (nt)
oligonucleotide (5’-GGTTCCACCAGCAACCCGCTA-3’)
as
the
template
and
a
14-nt
primer
(5’-
TAGCGGGTTGCTGG-3’), as shown in Figure 3. The
primer and template were designed in such a way that the
first incorporated base would be a T, so there are two possi-
ble scenarios in the extension, either fully extended product
or no extension at all. The latter case could arise from
either no incorporation of the modified nucleotide or the in-
ability to extend the sequence with the incorporation of this
modified nucleotide. The obtained DNA products were
studied using polyacrylamide gel electrophoresis (PAGE).
The primer was radio-labeled with 32P at the 5’-end using
g-32P-ATP and T4 kinase (lane 1, Figure 3). Negative control
without the Klenow fragment (lane 2, Figure 3) and without
dNTPs (lane 3, Figure 3) showed no full length DNA se-
quence. The shorter DNA in lane 3 could have resulted
from the 3’-5’ exonuclease activity of the Klenow frag-
ment.[8b] The positive control with the Klenow fragment and
natural dNTPs showed full length DNA sequence (lane 4,
Figure 3). Primer extension using B-TTP analogues 2–5
(lanes 5–8, Figure 3) also gave a full length DNA sequence,
which clearly indicated that these synthesized B-TTP ana-
logues 2–5 were recognized as a substrate by the poly-
merase, and incorporated into DNA.
PCR Investigation
Encouraged by the successful incorporation of B-TTP ana-
logues 2–5 into a short sequence by Klenow fragment-medi-
ated primer extension, we further investigated the PCR am-
plification of a longer DNA strand with a 90 bases template
that we used previously.[9] As can be seen from Figure 4, all
four analogues were successfully incorporated. Specifically,
very similar bands were found for the PCR products of B-
TTP analogues 2–5 (lanes 3–6, Figure 4, respectively, group
A) compared to those products using dTTP (lane 1, Figure 4
group A) and M-TTP (lane 7, Figure 4 group A). The slight-
ly reduced mobility of DNA with boronic acid incorporation
can be explained by the interaction of the boronic acid func-
tional group (a Lewis acid) with the PAGE gel. This can be
Chem. Asian J. 2011, 6, 2747 – 2752
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FULL PAPERS
Experimental Section
General
Chemicals were obtained from Aldrich and Acros, unless indicated other-
wise. For all reactions, analytical grade solvents were used. Anhydrous
solvents were used for all moisture-sensitive reactions. NMR data were
collected on a Bruker 400 MHz spectrophotometer. The chemical shifts
are relative to TMS as an internal standard for 1H NMR, and 85%
H3PO4 as an external reference for 31P NMR. Mass spectra were recorded
on a Waters Micromass LC-Q-TOF micro spectrometer or an ABI4800
MALDI-TOF/TOF mass spectrometer at Georgia State University, Mass
Spectrometry Facilities. HPLC conditions for the purification of B-TTP
analogues 2–5 were as follows: column: Agilent, semi-preparation
column; flow rate: 2.0 mLminÀ1
; solvents: A: 0.1m NH4HCO3, B:
MeOH; program: 0.02–15 min 25% (B%), 35–45 min, 100%, 55–65 min
25%, stop; temperature: 208C, detection wavelength: 280 nm.
Figure 4. Group A (analyzed with 15% PAGE) and B (15% denaturing
PAGE): PCR incorporation using dTTP (lane 1), B-TTP analogues 2–5
(lanes 3–6, respectively), or M-TTP (lane 7). Lane 2 is negative control
with no dTTP. Group C: (analyzed with 15% PAGE): PCR incorporation
using B-TTP analogues 2–5 (lanes 1, 3, 5, 7, respectively), DNA being
treated with H2O2 before analyzing (lanes 2, 4, 6, and 8 for B-TTP ana-
logues 2–5, respectively). All reactions were conducted using Taq poly-
merase (See Supporting Information for full gel pictures).
Synthesis of M(C)-TTP
The synthesis of M(C)-TTP followed modified procedure by Carell.[5a,b]
Compound M(C)-T[5a,b] (300 mg, 0.9 mmol, 1 eq) and proton sponge
(232 mg, 1.08 mmole, 1.2 eq) were dried in vacuo over P2O5 overnight
and then dissolved in anhydrous trimethylphosphate (2.5 mL) under ni-
trogen in an ice-bath. Then freshly distilled POCl3 (0.10 mL, 1.2 eq) dis-
solved in anhydrous trimethylphosphate (0.5 mL) was added dropwise
using a syringe with stirring. The reaction mixture was further stirred in
an ice-bath for 2 h and then a solution of bis-tri-n-butylammonium pyro-
phosphate (2.5 g, 5.27 mmole, 3.5 eq) and tri-n-butylamine (2.14 mL) in
3.0 mL of anhydrous DMF was added in one portion. The mixture was
stirred at room temperature for 10 min and then triethylammonium bi-
carbonate solution (0.1m, pH 8, 70 mL) was added. The reaction mixture
was stirred at room temperature for an additional hour, concentrated,
and then purified with a DEAE-Sephadex A-25 column using a linear
gradient of ammonium bicarbonate (0–0.6m). The portions eluted out
using 0.12–0.15m ammonium bicarbonate were collected. The process
was monitored by the UV absorbance at 290 nm. Lyophilization gave the
final product as a white powder (93 mg, 18%). 1H NMR (D2O): d=8.00
(s, 1H), 6.27 (t, J=6.8 Hz, 1H), 4.60 (s, 1H), 4.22 (m, 3H), 2.46 (t, J=
6.8 Hz, 2H), 2.41 (m, 2H), 2.36 (t, J=2.8 Hz, 1H), 2.27 (m, 2H),
1.68 ppm (m, 2H); 31P NMR (161 MHz, D2O): d=À6.1, À11.1,
À21.7 ppm; MS (-ESI) m/z: 571 ([MÀH]À).
further confirmed by the results of Group C, which repre-
sents the gel results of the PCR products after treating with
H2O2 (final concentration of 10 mm, 2 h). All B-TTP ana-
logues 2–5 (lanes 2, 4, 6, and 8, respectively, Figure 4 group
C) showed almost the same mobility. However, before treat-
ment, a different mobility was observed for B-TTP ana-
logues 2–5 (lanes 1, 3, 5, 7, respectively, Figure 4 Group C).
As an example, B-TTP analogue 4 shows the biggest differ-
ence in mobility (lane 5: before H2O2 treatment, and lane 6:
after H2O2 treatment, Figure 4 Group C). The most signifi-
cant evidence of the full incorporation of all the B-TTP ana-
logues came from the denaturing PAGE gel results (15%
denaturing PAGE containing 8m urea). A very clear single
band was observed in the case of B-TTP analogues 2–5
(lanes 3–6, Figure 4 Group B), compared to that of dTTP
(lane 1, Figure 4 group B).
Synthesis of B-TTP analogue 2
To a solution of M-TTP (5.0 mg, 0.0083 mmol, 1.0 eq) and 3-(azidometh-
yl)-phenyl-boronic acid (8.0 mg, 0.045 mmol, 5.4 eq) in 120 mL of a mixed
solvent (H2O/DMF/EtOH=1:2:1) was added 50 mL of a solution of
TBTA (2.8 mg, 0.0052 mmole, 0.60 eq) and CuBr (0.2 mg, 0.0026 mmole,
0.30 eq) in 100 mL DMF. Then the mixture was stirred vigorously at room
temperature for 3 h and centrifuged. Supernatant was removed, and the
remaining was washed twice with 100 mm NH4HCO3 buffer (0.7 mL).
The combined washings and supernatant were purified by HPLC to give
a white powder after lyophilization (1.9 mg, 31%). 1H NMR (D2O): d=
7.68 (s, 1H), 7.66 (s, 1H), 7.00 (t, J=8.4 Hz, 1H), 6.53 (d, J=8.4 Hz,
1H), 6.34 (s, 1H), 6.05 (t, J=6.8 Hz, 1H), 5.33 (s, 2H), 4.34 (m, 1H),
4.00 (m, 3H), 3.92 (s, 2H), 2.91 (t, J=6.4 Hz, 2H), 2.45 (t, J=6.4 Hz,
2H), 2.12 ppm (m, 2H); 31P NMR (161 MHz, D2O): d=À5.9, À10.8,
À19.2 ppm; MS (-ESI) m/z: 733 ([MÀBOHÀH2OÀH]À).
Conclusions
Four boronic acid-modified TTP analogues were successfully
synthesized. Two of them were synthesized using an im-
proved procedure, which uses 1,7-octadiyne as a linker for
both the Sonogashira coupling with thymidine and the
CuAAC tethering of the boronic acid moiety. All four bor-
onic acid-modified TTP analogues were characterized by
1H NMR, 31P NMR, and MS, and purities were confirmed
by HPLC. Moreover, these analogues were successfully in-
corporated into DNA, suggesting that linker differences and
the structural features of the boronic acid part do not have
much bearing on polymerase-mediated incorporation. The
newly developed synthetic method also paves the way for
the preparation of a large number of boronic acid-modified
TTP with a diverse set of structural features for future appli-
cations such as aptamer selection.
Synthesis of B-TTP Analogue 3
To a solution of M-TTP (5.0 mg, 0.0083 mmol, 1.0 eq) and 2-(azidometh-
yl)-phenyl-boronic acid (5.1 mg, 0.029 mmol, 3.4 eq) in 120 mL of a mixed
solvent (H2O/DMF/EtOH=1:2:1) was added 45 mL of a solution of
TBTA (2.8 mg, 0.0052 mmole, 0.62 eq) and CuBr (0.4 mg, 0.0028 mmole,
0.34 eq) in 90 mL DMF. Then the mixture was stirred vigorously at room
temperature for 3 h and centrifuged. Supernatant was removed, and the
remaining was washed twice with 100 mm NH4HCO3 buffer (0.7 mL).
The combined washings and supernatant were purified by HPLC to give
a white powder after lyophilization (2.0 mg, 31%). 1H NMR (D2O): d=
7.69 (s, 1H), 7.65 (s, 1H), 7.01 (m, 1H), 6.57 (m, 1H), 6.37 (m, 2H), 6.04
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Incorporation of Click-Modified Boronic Acid–TTP Analogues
(t, J=6.8 Hz, 1H), 5.61 (s, 2H), 4.35 (m, 1H), 4.04 (m, 3H), 3.92 (s, 2H),
2.92 (t, J=6.4 Hz, 2H), 2.46 (t, J=6.4 Hz, 2H), 2.11 ppm (m, 2H);
31P NMR (161 MHz, D2O): d=À5.1, À10.0, À18.5 ppm; MS (-ESI) m/z:
669 ([MÀBOHÀPO3HÀH]À).
PCR Incorporation using B-TTP Analogues
PCR was performed on an Eppendorf Mastercycler thermal cycler with
ethidium bromide fluorescent imaging. Taq DNA polymerase was pur-
chased from New England Biolabs. Reaction buffer was used as provided
by the vendor. 50 mL reaction mixture contains template (90-mer single-
strand DNA pool 5’-CCTTCGTTGTCTGCCTTCGT-50N-ACCCTTCA-
GAATTCGCACCA-3’, with a final concentration 10 nm, where 50N
Synthesis of B-TTP Analogue 4
To a solution of M(C)-TTP (5.0 mg, 0.0087 mmol, 1.0 eq) and 3-(azido-
methyl)-phenyl-boronic acid (8.0 mg, 0.045 mmol, 5.2 eq) in 120 mL of a
mixed solvent (H2O/DMF/EtOH=1:2:1) was added 50 mL of a solution
of TBTA (2.8 mg, 0.0052 mmol, 0.60 eq) and CuBr (0.4 mg, 0.0026 mmol,
0.30 eq) in 100 mL DMF. The mixture was stirred vigorously at room tem-
perature for 3 h and centrifuged. Supernatant was removed, and the re-
maining was washed twice with 100 mm NH4HCO3 buffer (0.7 mL). The
combined washings and supernatant were purified by HPLC to give a
white powder after lyophilization (1.9 mg, 29%). 1H NMR (D2O): d=
7.86 (s, 2H), 7.63 (d, J=7.2 Hz, 1H), 7.55 (s, 1H), 7.36 (t, J=7.2 Hz,
1H), 7.24 (d, J=7.6 Hz, 1H), 6.25 (t, 1H), 5.59 (s, 1H), 4.58 (m, 2H),
4.22 (m, 3H), 2.76 (t, J=6.8 Hz, 2H), 2.41 (m, 4H), 1.79 (t, J=6.4 Hz,
2H), 1.57 ppm (t, J=6.4 Hz, 2H); 31P NMR (161 MHz, D2O): d=À5.4,
À10.3, À18.7 ppm; MS (-ESI) m/z: 355.6 ([MÀ2H2OÀ2H]2À).
stands for 50 randomized positions), primer
TGGTGCGAATTCTGAAGGGT-3’), primer
1
(100 mm, 5’-
(1 mm, 5’-
2
CCTTCGTTGTCTGCCTTCGT-3’), dATP, dCTP, dGTP, dTTP, or one
of the B-TTP analogues or MTTP (each with a final concentration of
200 mm), 1ꢁreaction buffer, and Taq polymerase (0.5 U). Reaction ther-
mocycling is composed of initial denaturation (958C 2 min), 30 cycles of
958C 20 s, 488C 20 s, and 728C 30 s, and final extension (728C 10 min)
then held at 48C. Reaction product was purified by washing with H2O in
a Millipore Amicon Ultra 10 kDa spin column. The product was then an-
alyzed with 15% PAGE by loading with 1ꢁgel-loading dye (2.5% Ficoll
400, 11 mm EDTA, 3.3 mm Tris-HCl, 0.017% SDS, 0.015% bromophenol
blue at pH 8.0). Denaturing gel analysis was performed by denaturing
the purified PCR product at 958C for 2 min, followed by addition of 1ꢁ
loading dye with 8m urea at 708C and kept for 2 min. The sample was
then loaded onto 15% denaturing PAGE containing 8m urea. One por-
tion of purified PCR product was treated with H2O2 (final concentration
10 mm) at RT for 2 h before loading onto 15% PAGE.
Synthesis of B-TTP Analogue 5
To a solution of M(C)-TTP (5.0 mg, 0.0087 mmole, 1.0 eq) and 2-(azido-
methyl)-phenyl-boronic acid (8.0 mg, 0.045 mmol, 5.2 eq) in 120 mL of a
mixed solvent (H2O/DMF/EtOH=1:2:1) was added 50 mL of a solution
of TBTA (2.8 mg, 0.0052 mmole, 0.60 eq) and CuBr (0.4 mg,
0.0026 mmole, 0.30 eq) in 100 mL DMF. The mixture was stirred vigorous-
ly at room temperature for 3 h and centrifuged. Supernatant was re-
moved, and then the remaining was washed twice with 100 mm
NH4HCO3 buffer (0.7 mL). The combined washings and supernatant
were purified by HPLC to give a white powder after lyophilization
Acknowledgements
Financial support from the NIH (GM086925 and GM084933) and the
Molecular Basis of Disease program and University Fellowship Program
at Georgia State University is gratefully acknowledged.
1
(0.9 mg, 14%). H NMR (D2O): d=7.70 (s, 1H), 7.60 (s, 1H), 7.41 (d, J=
6.4 Hz, 1H), 7.24 (m, 1H), 7.09 (d, J=6.4 Hz, 1H), 6.74 (m, 1H), 6.07 (t,
J=6.4 Hz, 1H), 5.53 (s, 2H), 4.41 (m, 1H), 4.04 (m, 3H), 2.56 (t, J=
6.8 Hz, 2H), 2.20 (m, 4H), 1.59 (t, J=7.6 Hz, 2H), 1.37 ppm (t, J=
7.6 Hz, 2H); 31P NMR (161 MHz, D2O): d=À5.1, À10.0, À18.5 ppm; MS
(-ESI) m/z: 355.6 ([MÀ2H2OÀ2H]2À).
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PAGE Analysis of Klenow-Fragment-Catalyzed Primer Extension
The mixture of 14-nt primer DNA (50 mm), T4 polynucleotide kinase
(0.5 unitsmLÀ1, Biolabs, Inc.) and g-32P-ATP (0.6 mL, from Perkin–Elmer
Corp.) in 12 mL T4 kinase buffer solution was incubated at 378C for 1 h.
The harvested 32P-labeled DNA was then purified using Microcon YM-3
centrifugal filter (Millipore Corp.) to remove low molecular weight mole-
cules. Using
a similar primer extension protocol as previously de-
scribed,[8] the 32P-labeled primer alone, reaction mixture without enzyme,
reaction mixture without dNTPs, reaction mixture with both enzyme and
natural dNTPs, and reaction mixture with B-TTP analogues 2–5 together
with the other 3 dNTPs as well as enzyme were incubated at 378C for
1 h. After reaction, the mixtures were quenched with 2ꢁDNA loading
dye. 3 mL of samples from each reaction was taken and run on 15%
PAGE at 300 V for 3 h. After being isolated, fixed, and dried, the gel was
developed using autoradiography (overnight) to obtain the film.
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Primer Extension using the Klenow Fragment for MALDI-TOF-MS
Studies
10 mL of 21-nt template (100 mm), 15 mL of 14-nt primer (100 mm), 1 mL of
Klenow (5 unitsmLÀ1), 4 mL of dNTPs (0.2 mm each), 5 mL of 10ꢁNEB
buffer 2, 15 mL of deionized water in a total volume of 50 mL of solution
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(100 mm), 15 mL of 14-nt primer (100 mm), 1 mL of Klenow (5 unitsmLÀ1),
4 mL of B-TTP analogue 2 (2.5 mm), 4 mL of three other dNTPs (0.2 mm
each), 5 mL of 10ꢁNEB buffer 2, and 11 mL of de-ionized water in a total
volume of 50 mL of solution was prepared for the reaction by using B-
TTP analogue 2. The prepared solutions were then incubated at 378C for
1 h. After further purification using Microcon YM-3 centrifugal filter
(Millipore Corp.) to remove dNTPs and other low molecular weight mol-
ecules, the harvested DNA was directly sent for MALDI analysis.
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Received: March 3, 2011
Published online: September 2, 2011
2752
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ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Asian J. 2011, 6, 2747 – 2752