An unnatural hydrophobic base pair with shape complementarity between pyrrole-2-carbaldehyde and 9-methylimidazo[(4,5)-b]pyridine

Article abstract of DOI:10.1021/ja028806h

An unnatural hydrophobic base, pyrrole-2-carbaldehyde (denoted as Pa), was developed as a specific pairing partner of 9-methylimidazo[(4,5)-b]pyridine (Q). The Q base is known to pair with 2,4-difluorotoluene (F) as an isostere of the A-T pair, and F also

Full text of DOI:10.1021/ja028806h

Published on Web 04/11/2003
An Unnatural Hydrophobic Base Pair with Shape
Complementarity between Pyrrole-2-carbaldehyde and
9-Methylimidazo[(4,5)-b]pyridine
Tsuneo Mitsui,†,‡,§ Aya Kitamura,‡,# Michiko Kimoto,‡,# Taiko To,‡,# Akira Sato,†,#
Ichiro Hirao,*,†,‡,§ and Shigeyuki Yokoyama*,†,‡,#
Contribution from the Yokoyama CytoLogic Project, ERATO, JST, c/o RIKEN, 2-1 Hirosawa,
Wako, Saitama 351-0198, Japan, Protein Synthesis Technology Team, RIKEN Genomic Sciences
Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama, Kanagawa 230-0045, Japan, Research Center
for AdVanced Science and Technology, The UniVersity of Tokyo, 4-6-1 Komaba,
Meguro-ku, Tokyo 153-8904, Japan, and Department of Biophysics and Biochemistry, Graduate
School of Science, The UniVersity of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
Received October 4, 2002; E-mail: ihirao@postman.riken.go.jp and yokoyama@biochem.s.u-tokyo.ac.jp
Abstract: An unnatural hydrophobic base, pyrrole-2-carbaldehyde (denoted as Pa), was developed as a
specific pairing partner of 9-methylimidazo[(4,5)-b]pyridine (Q). The Q base is known to pair with 2,4-
difluorotoluene (F) as an isostere of the A-T pair, and F also pairs with A efficiently in replication. In
contrast, the Q-Pa pair showed specific selectivity in replication, and the five-membered-ring base Pa
paired efficiently with Q but paired poorly with A. In addition, the interaction of Pa with DNA polymerases
was superior, in comparison to that of F. The aldehyde group of Pa was recognized well by the Klenow
fragment of Escherichia coli DNA polymerase I and the reverse transcriptase of Avian myeloblastosis virus.
The structural features of the Q-Pa pair in a DNA duplex were analyzed by NMR, showing the shape
complementarity of the Pa fitting with Q. The structurally unique base Pa provides valuable information for
the development of unnatural base pairs toward the expansion of the genetic alphabet.
Introduction
ylbenzimidazole (Z) and 2,4-difluorotoluene (F), and 9-meth-
ylimidazo[(4,5)-b]pyridine (Q) and F. The nucleotides of Z and
Creating unnatural base pairs that have exclusive selectivity
in replication, transcription, and translation would lead to great
advancements in the bioengineering of nucleic acids and
proteins.1-3 The expansion of the genetic codes permits the site-
specific incorporation of amino acid analogues into proteins in
vitro and in vivo. In addition, the introduction of unnatural
nucleotide components into nucleic acids facilitates their
increased functionality.
For the development of unnatural base pairs, Benner and
colleagues showed the importance of the hydrogen-bonding
patterns between pairing bases by their studies of unnatural base
pairs, such as isoG and isoC.4-6 Recently, Kool and colleagues
synthesized non-hydrogen-bonded base pairs, such as 4-meth-
F or Q and F are enzymatically incorporated into DNA opposite
each other.7-10 From this research, they developed two con-
cepts: the importance of the shape complementarity between
pairing bases and the potential of hydrophobic bases as artificial
base pairs.4 By combining the concepts of hydrogen-bonding
patterns4-6 and shape complementarity,9-11 we developed
unnatural base pairs between 2-amino-6-dimethylaminopurine
(x) and pyridin-2-one (y),12,13 and between 2-amino-6-(2-
thienyl)purine (s) and y.14,15 These unnatural base pairs showed
high specificity in transcription13,15 and were applied to coupled
transcription and translation for the site-specific incorporation
of unnatural amino acids into proteins.15 On the other hand,
the concept of a hydrophobic base pair was applied to the
creation of unnatural base pairs for the expansion of the genetic
† Yokoyama CytoLogic Project, ERATO, JST.
‡ Protein Synthesis Technology Team, RIKEN Genomic Sciences Center.
§ Research Center for Advanced Science and Technology, The University
of Tokyo.
(7) Morales, J. C.; Kool, E. T. J. Am. Chem. Soc. 1999, 121, 2323-2324.
(8) Kool, E. T.; Morales, J. C.; Guckian, K. M. Angew. Chem., Int. Ed. 2000,
39, 990-1009.
(9) Morales, J. C.; Kool, E. T. Nat. Struct. Biol. 1998, 5, 950-954.
(10) Matray, T. J.; Kool, E. T. Nature 1999, 399, 704-708.
(11) Rappaport, H. P. Biochemistry 1993, 32, 3047-3057.
(12) Ishikawa, M.; Hirao, I.; Yokoyama, S. Tetrahedron Lett. 2000, 41, 3931-
3934.
# Department of Biophysics and Biochemistry, The University of Tokyo.
(1) Benner, S. A.; Burgstaller, P.; Battersby, T. R.; Jurczyk, S. The RNA World,
2nd ed.; Gesteland, R. F., Cech, T. R., Atkins, J. F., Eds.; Cold Spring
Harbor Laboratory Press; Cold Spring Harbor, NY, 1999; pp 163-
181.
(2) Kool, E. T. Curr. Opin. Chem. Biol. 2000, 4, 602-608.
(3) Service, R. F. Science 2000, 289, 232-235.
(13) Ohtsuki, T.; Kimoto, M.; Ishikawa, M.; Mitsui, T.; Hirao, I.; Yokoyama,
S. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 4922-4925.
(4) Switzer, C.; Moroney, S. E.; Benner, S. A. J. Am. Chem. Soc. 1989, 111,
8322-8323.
(14) Fujiwara, T.; Kimoto, M.; Sugiyama, H.; Hirao, I.; Yokoyama, S. Bioorg.
Med. Chem. Lett. 2001, 11, 2221-2223.
(5) Piccirilli, J. A.; Krauch, T.; Moroney, S. E.; Benner, S. A. Nature 1990,
343, 33-37.
(15) Hirao, I.; Ohtsuki, T.; Fujiwara, T.; Mitsui, T.; Yokogawa, T.; Okuni, T.;
Nakayama, H.; Takio, K.; Yabuki, T.; Kigawa, T.; Kodama, K.; Yokogawa,
T.; Nishikawa, K.; Yokoyama, S. Nat. Biotechnol. 2002, 20, 177-182.
(6) Horlacher, J.; Hottiger, M.; Podust, V. N.; Hu¨bscher, U.; Benner, S. A.
Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 6329-6333.
9
5298
J. AM. CHEM. SOC. 2003, 125, 5298-5307
10.1021/ja028806h CCC: $25.00 © 2003 American Chemical Society

Hydrophobic Base Pairs with Shape Complementarity
A R T I C L E S
has a nitrogen at the site corresponding to position 3 of the
natural purine bases that can interact with polymerases. Thus,
the development of an unnatural base, which is replaced by F
and specifically pairs with Q, would lead to the creation of an
unnatural base pair with exclusive selectivity in replication.
To address these problems of the F base, we considered the
stringent shape complementarity for the pairing partner of Q
and the effective interactions of the partner base with DNA
polymerases, and thus the hydrophobic pyrrole-2-carbaldehyde
(Pa) was newly designed (structure D in Figure 1). The five-
membered ring of Pa would avoid the steric collision with Q,
and the aldehyde group of Pa could interact with polymerases.
In addition, the fitting between A and Pa might be inferior to
that between A and F. This is because the stacking stability of
Pa might be weaker than that of F, and, thus, the A-Pa pair
would be destabilized by the water solvation of A8 (structure E
in Figure 1). We synthesized the nucleotides of Pa, examined
the efficiency of Q-Pa pairing in replication, and analyzed the
structural features of the Q-Pa pair in a DNA duplex.
Experimental Section
Sample Preparation. The nucleoside triphosphates and phosphora-
midites of F and Q were synthesized with a slight modification of the
method described in the literature.7 The phosphoramidites of isoG and
isoC (5-methylisocytosine) were purchased from GLEN RESEARCH
(Sterling, VA). DNA fragments were synthesized with an Applied
Biosystems 392 DNA synthesizer (Applied Biosystems, Foster City,
CA) and were purified either by HPLC, using SynChropak RPP
(Eichrom Technologies, Darien, IL), or by gel-electrophoresis after
deprotection.
Synthesis of 1-(2′-Deoxy-3′,5′-di-O-toluoyl-â-D-ribofuranosyl)-
pyrrole-2-carbaldehyde. To a solution of pyrrole-2-carbaldehyde (368
mg, 4.4 mmol) in CH3CN (56 mL) was added NaH (219 mg, 60%
dispersion in mineral oil, 1.4 equiv), and the mixture was stirred at
room temperature for 1 h. A quantity of 1-chloro-2-deoxy-3,5-di-O-
toluoyl-R-D-erythropentofuranose (1.5 g, 4.4 mmol) was then added
to the solution. The reaction mixture was stirred at room temperature
for 1 h and was partitioned with EtOAc and H2O. The organic layer
was washed with H2O, dried with Na2SO4, and evaporated in vacuo.
The product (1.2 g, 60%) was purified from the residue by silica gel
column chromatography (1% MeOH in CH2Cl2). 1H NMR (270 MHz,
CDCl3) δ 2.33-2.44 (m, 7H, H2′, CH3 × 2), 2.89 (m, 1H, H2′′), 4.55-
4.67 (m, 3H, H4′, H5′, H5′′), 5.56 (m, 1H, H3′), 6.23 (t, 1H, H1′, J )
3.6 Hz), 6.96-7.01 (m, 2H, pyrrole), 7.23 (dd, 4H, phenyl, J ) 8.2,
19.4 Hz), 7.45 (s, 1H, pyrrole), 7.91 (dd, 4H, phenyl, J ) 8.2, 19.4
Hz), 9.52 (s, 1H, -CHO); thin-layer chromatography (TLC) Rf ) 0.34
(CH2Cl2:MeOH ) 100:1 v/v). High-resolution mass spectroscopy
(HRMS) (FAB, 3-NBA matrix) for C26H26NO6 (M + 1): calcd,
448.1760; found, 448.1751.
Figure 1. Base-pair structures: (A) the Q-F base pair, (B) the natural
A-T pair, (C) a noncognate A-F pair, (D) the novel base Pa pairing with
Q, and (E) a noncognate A-Pa pair. In these structures, R denotes ribose.
alphabet by the Romesberg and Schultz groups.16-20 These
hydrophobic base pairs are the potential candidates for unnatural
base pairs at present.
To expand the development of unnatural base pairs, we
focused on the hydrophobic Z-F and Q-F pairs. One of the
problems of these base pairs is their poor exclusivity, because
the shape-fitted Z-F and Q-F pairs are isosteres of the A-T
pair, and thus efficient noncognate base pairings, especially
between A and F, appear in replication7,21,22 (see structures A-C
in Figure 1). The shape of F mimics that of T, and F fits well
with A. The A-F pair is much more efficient than the other
noncognate Z-T and Q-T pairs. This may be because the H3
of T clashes with the hydrogens of Z and Q on the pairing
surface. Similarly, the hydrogen of F, corresponding to the H3
of pyrimidines, may clash with the hydrogens of Z and Q
(structure A in Figure 1). Actually, the geometry of the Z-F
pair is slightly distorted in the DNA duplex, compared to that
of the A-T pair.23 Thus, the A-F pair is the most shape-fitted
base pair among the cognate and noncognate pairs involving
Z, Q, and F. Another problem is that F is not recognized by
some polymerases, because it lacks the 2-keto group of the
natural pyrimidines.21,22 In this regard, the recognition of Z by
DNA polymerases was improved by replacing it with Q,7 which
Synthesis of 1-(2′-Deoxy-â-D-ribofuranosyl)-pyrrole-2-carbalde-
hyde. A quantity of 1-(2′-deoxy-3′,5′-di-O-toluoyl-â-D-ribofuranosyl)-
pyrrole-2-carbaldehyde (100 mg, 0.22 mmol) was poured into saturated
ammonia in methanol (4.0 mL), and the solution was stirred overnight
at room temperature. The reaction mixture was evaporated in vacuo
and separated with H2O and EtOAc, and then the water layer was
evaporated in vacuo. The product (35 mg, 74%) was purified by
reversed-phase HPLC (Waters Microbond Sphere model C18, with a
(16) McMinn, D. L.; Ogawa, A. K.; Wu, Y.; Liu, J.; Schultz, P. G.; Romesberg,
F. E. J. Am. Chem. Soc. 1999, 121, 11585-11586.
(17) Ogawa, A. K.; Wu, Y.; McMinn, D. L.; Liu, J.; Schultz, P. G.; Romesberg,
F. E. J. Am. Chem. Soc. 2000, 122, 3274-3287.
1
gradient from 5% to 50% (10 min) CH3CN in H2O). H NMR (270
(18) Wu, Y.; Ogawa, A. K.; Berger, M.; McMinn, D. L.; Schultz, P. G.;
Romesberg, F. E. J. Am. Chem. Soc. 2000, 122, 7621-7632.
(19) Ogawa, A. K.; Wu, Y.; Berger, M.; Schultz, P. G.; Romesberg, F. E. J.
Am. Chem. Soc. 2000, 122, 8803-8804.
MHz, DMSO-d6) δ 2.13 (m, 1H, H2′â), 2.26 (m, 1H, H2′′R), 3.54 (m,
2H, H5′, H5′′), 3.79 (m, 1H, H4′), 4.24 (m, 1H, H3′), 4.95 (t, 1H,
5′-OH, J ) 5.3 Hz, D2O exchange), 5.23 (d, 1H, 3′-OH, J ) 4.1 Hz,
D2O exchange), 6.28 (t, 1H, pyrrole H4), 6.73 (t, 1H, H1′, J ) 6.4
Hz), 7.06 (dd, 1H, pyrrole H3, J ) 1.6, 3.8 Hz), 7.70 (bs, 1H, pyrrole
H5), 9.50 (s, 1H, -CHO). 13C NMR (68 MHz, DMSO-d6) δ 41.71,
41.83, 41.96, 61.28, 61.34, 61.41, 70.09, 85.74, 85.84, 87.33, 110.02,
(20) Tae, E. L.; Wu, Y.; Xia, G.; Schultz, P. G.; Romesberg, F. E. J. Am. Chem.
Soc. 2001, 123, 7439-7440.
(21) Morales, J. C.; Kool, E. T. J. Am. Chem. Soc. 2000, 122, 1001-1007.
(22) Morales, J. C.; Kool, E. T. Biochemistry 2000, 39, 12979-12988.
(23) Guckian, K. M.; Krugh, T. R.; Kool, E. T. J. Am. Chem. Soc. 2000, 122,
6841-6847.
9
J. AM. CHEM. SOC. VOL. 125, NO. 18, 2003 5299

A R T I C L E S
Mitsui et al.
125.16, 127.67, 127.77, 130.86, 179.21, 179.28. Electrospray ioniza-
tion-mass spectroscopy (ESI-MS) for C10H13NO4: calcd, 210.08
[M-H]-; found, 210.05 [M-H]-. UV-vis (in EtOH): λmax ) 288
nm (ꢀ ) 11 230), 260 nm (ꢀ ) 5610); λmin ) 220 nm (ꢀ ) 670). TLC
Rf ) 0.21 (CH2Cl2:MeOH ) 20:1 v/v). The anomeric configuration of
1-(2′-deoxy-â-D-ribofuranosyl)-pyrrole-2-carbaldehyde was confirmed
using nuclear Overhauser effects (NOEs) (see the Supporting Informa-
tion). HRMS (FAB, 3-NBA matrix) for C10H14NO4 (M + 1): calcd,
212.0923; found, 212.0922.
H2′′), 3.02 (q, 18H, -CH2CH3, J ) 7.3 Hz), 3.96-4.04 (m, 3H, H4′,
H5′, H5′′), 4.51 (m, 1H, H3′), 6.28 (dd, 1H, pyrrole H4, J ) 2.8 Hz,
4.0 Hz), 6.77 (t, 1H, H1′, J ) 6.6 Hz), 7.05 (dd, 1H, pyrrole H3, J )
1.7 Hz, 4.0 Hz), 7.59 (s, 1H, pyrrole H5), 9.25 (s, 1H, -CHO). 31P
NMR (109 MHz, D2O) δ -22.91 (t, 1H, J ) 20.1 Hz), -10.96 (d, 1H,
J ) 20.1 Hz), -10.40 (d, 1H, J ) 19.5 Hz); ESI-MS for C10H16-
NO13P3: calcd, 449.98 [M-H]-; found, 449.80 [M-H]-.
Thermal Denaturation. The concentration of DNA fragments was
determined from each hypochromicity, obtained by the complete
degradation of the fragments with nuclease P1. The absorbance at 260
nm of the DNA fragments was monitored as a function of temperature
(15-65 °C) on a Beckman model DU650 spectrophotometer. The
duplexes of 5′-GGTAACNATGCG and 5′-CGCATN′GTTACC (N )
Q, A, or G, and N′ ) Pa or T) were dissolved in 10 mM sodium
phosphate (pH 7.0), 100 mM NaCl, and 0.1 mM EDTA to give a duplex
concentration of 5 mM. Melting temperature (Tm) values were calculated
by the first derivatives of the melting curves.
Synthesis of 5′-O-Dimethoxytrityl-1-(2′-deoxy-â-D-ribofuranosyl)-
pyrrole-2-carbaldehyde. A quantity of 1-(2′-deoxy-â-D-ribofuranosyl)-
pyrrole-2-carbaldehyde (100 mg, 0.47 mmol) was coevaporated with
dry pyridine three times. The residue was dissolved in pyridine (4.7
mL) with 4,4-dimethoxytrityl chloride (160 mg, 0.47 mmol), and the
solution was stirred at room temperature for 4 h. Water was then added
to the solution and the product was extracted with EtOAc. The organic
layer was washed with 5% NaHCO3 three times, dried with Na2SO4,
and evaporated in vacuo. The product was purified by silica gel column
chromatography (1% MeOH in CH2Cl2) to give the dimethoxytrityl
Steady-State Kinetics. Steady-state kinetics for single-nucleotide
insertions were performed according to the literature.25,26 Primers were
5′-labeled, using [γ-32P]ATP and T4 polynucleotide kinase. Primer-
template duplexes (10 µM) were annealed in a buffer containing 100
mM tris-HCl (pH 7.5), 20 mM MgCl2, 2 mM DTT, and 0.1 mg/mL
bovine serum albumin by heating at 95 °C and slow cooling to 4 °C.
The duplex solution (5 µL) was mixed with 2 µL of a solution
containing the exonuclease-deficient Klenow fragment (Amersham
USB, Cleveland, OH) diluted in a buffer containing 50 mM potassium
phosphate (pH 7.0), 1 mM DTT, and 50% glycerol, and was incubated
at 37 °C for more than 2 min. Reactions were initiated by adding 3 µL
of a dNTP solution to the DNA-enzyme mixture at 37 °C. The amount
of polymerase used (3-50 nM), the reaction time (1-22 min), and
the gradient concentration of dNTP (0.6-2100 µM) were adjusted to
give reaction extents of 25% or less. Reactions were quenched by adding
10 µL of a dye solution containing 89 mM trisborate, 2 mM EDTA,
and 10 M urea, and the mixtures were immediately heated at 75 °C for
3 min. The products were analyzed on a 15%-20% polyacrylamide
gel containing 7 M urea. The reaction extents were measured with a
bio-imaging analyzer (Fuji model BAS 2500). Relative velocities (V0)
were calculated as the extents of the reaction divided by the reaction
time and were normalized to the enzyme concentration (20 nM) for
the various enzyme concentrations used. The kinetic parameters (Km
and Vmax) were obtained from Hanes-Woolf plots of [dNTP]/V0 against
[dNTP]. Each parameter was averaged from 3-12 data sets.
Primer Extension Reaction. The 5′-labeled primer was annealed
to the template in an annealing buffer, by heating at 95 °C and slow
cooling to 4 °C. The duplex solution (5 µL) was mixed with 2 µL of
solution A containing dNTP substrates, and polymerase reactions were
started by adding 3 µL of solution B containing the polymerase. The
reaction mixture was incubated at 37 °C and was terminated by adding
10 µL of the dye solution and heating at 75 °C for 3 min. The products
were analyzed on a 15% polyacrylamide gel containing 7 M urea.
(A) KF Reactions. The annealing buffer contained 20 mM tris-
HCl (pH 7.5), 14 mM MgCl2, and 0.2 mM DTT. Solution A contained
50 µM dNTPs, and solution B contained the exonuclease-proficient
Klenow fragment (TaKaRa, Tokyo) diluted in distilled water. The
concentrations used for primer extension were as follows: primer-
template, 200 nM; KF, 0.1 unit/µL; and dNTPs, 10 µM each; the
incubation time for each was 5 min. Unit definition: One unit
incorporates 10 nmol of total nucleotides into acid-insoluble products
in 30 min at 37 °C using poly d(A-T) as the template-primer.
(B) Reverse Transcriptase of Avian Myeloblastosis Virus (AMV-
RT) Reactions. Solution A contained 500 µM dNTPs diluted in an
annealing buffer [50 mM tris-HCl (pH 8.3), 10 mM MgCl2, 100 mM
KCl, and 4 mM DTT], and solution B contained the reverse transcriptase
1
derivative (240 mg, 99%). H NMR (270 MHz, CDCl3) δ 2.27 (m,
1H, H2′), 2.53 (m, 1H, H2′′), 3.41 (d, 2H, H5′, H5′′, J ) 4.3 Hz), 3.78
(s, 6H, -OCH3 × 2), 4.02 (m, 1H, H4′), 4.44 (m, 1H, H3′), 6.15 (m,
1H, pyrrole H4), 6.79-6.84 (m, 5H, DMTr, H1′), 6.95 (m, 1H, pyrrole
H3), 7.21-7.46 (m, 10H, pyrrole H5, DMTr), 9.50 (s, 1H, -CHO).
TLC Rf ) 0.51 (CH2Cl2:MeOH ) 20:1 v/v). HRMS (FAB, 3-NBA
matrix) for C31H32NO6 (M + 1): calcd, 514.2230; found, 514.2234.
Synthesis of 5′-O-Dimethoxytrityl-1-(2′-deoxy-â-D-ribofuranosyl)-
pyrrole-2-carbaldehyde-3′-phosphoramidite. A quantity of 5′-O-
dimethoxytrityl-1-(2′-deoxy-â-D-ribofuranosyl)-pyrrole-2-carbalde-
hyde (103 mg, 0.20 mmol) was coevaporated with pyridine and
tetrahydrofuran (THF) three times each and was dissolved in THF (1.0
mL) with diisopropylethylamine (39 µL, 1.1 equiv) and 2-cyanoethyl-
N,N-diisopropylamino-chlorophosphoramidite (50 µL, 1.1 equiv). The
reaction mixture was stirred at room temperature for 2 h, and then
MeOH (50 µL) was added. The solution was diluted with EtOAc/
triethylamine (EtOAc/TEA) (10 mL, 20:1 v/v) and then was washed
with 5% NaHCO3 and saturated NaCl three times each. The organic
layer was dried with Na2SO4 and evaporated in vacuo. The residue
was purified by silica gel column chromatography (3:2 (v/v) hexane:
CH2Cl2 ratio, containing 2% TEA) to give the amidite (147 mg, 100%).
1H NMR (270 MHz, CDCl3) δ 1.01-1.16 (m, 12H, -CH(CH3)2 × 2),
2.23 (m, 1H, H2′), 2.39 (t, 1H, -CH2-), 2.58-2.63 (m, 2H, H2′′,
-CH2-), 3.25-3.61 (m, 5H, H5′, H5′′, -CH(CH3)2 × 2, -CH2-),
3.77 (s, 7H, -OCH3 × 2, -CH2-), 4.16 (m, 1H, H4′), 4.54 (m, 1H,
H3′), 6.14 (m, 1H, pyrrole H4), 6.78-6.86 (m, 5H, DMTr, H1′), 6.94
(m, 1H, pyrrole H3), 7.16-7.44 (m, 9H, DMTr), 7.48 and 7.56 (dbs,
1H, pyrrole H5), 9.51 (s, 1H, -CHO). 31P NMR (109 MHz, CDCl3) δ
148.90, 149.63 (diastereoisomers). TLC Rf ) 0.32 (hexane:CH2Cl2 )
2:3 v/v, containing 2% TEA). HRMS (FAB, 3-NBA matrix) for
C40H49N3O7P (M + 1): calcd, 714.3308; found, 714.3298.
Synthesis of 1-(2′-Deoxy-â-D-ribofuranosyl)-pyrrole-2-carbalde-
hyde-5′-triphosphate. To a solution of 1-(2′-deoxy-â-D-ribofuranosyl)-
pyrrole-2-carbaldehyde (22 mg, 0.1 mmol) and a proton sponge (33
mg, 0.15 mmol) in trimethyl phosphate (500 µL) was added POCl3
(13 µL, 1.3 equiv) at 0 °C.24 The reaction mixture was stirred at 0 °C
for 1.5 h. Tri-n-butylamine (119 µL, 5.0 equiv) was added to the
reaction mixture, followed by 0.5 M bis(tributylammonium)pyrophos-
phate in a DMF solution (1.0 mL, 5.0 equiv). After 30 min, the reaction
was quenched by the addition of 0.5 M triethylammonium bicarbonate
(TEAB, 500 µL). The resulting crude solution was purified by DEAE
Sephadex A-25 column chromatography (1.5 cm × 30 cm, eluted by
a linear gradient of 50 mM to 1 M TEAB), and then by C18-HPLC
(Synchropak RPP, Eichrom Technologies, eluted by a gradient of 0%-
1
30% CH3CN in 100 mM triethylammonium acetate). H NMR (270
MHz, D2O) δ 1.10 (t, 27H, -CH2CH3, J ) 7.3 Hz), 2.34 (m, 2H, H2′,
(24) Kova´cs, T.; O¨ tvo¨s, L. Tetrahedron Lett. 1988, 29, 4525-4528.
(25) Petruska, J.; Goodman, M. F.; Boosalis, M. S.; Sowers, L. C.; Cheong, C.;
Tinoco, I. Proc. Natl. Acad. Sci. U.S.A. 1988, 85, 6252-6256.
(26) Goodman, M. F.; Creighton, S.; Bloom, L. B.; Petruska, J. Crit. ReV.
Biochem. Mol. Biol. 1993, 28, 83-126.
9
5300 J. AM. CHEM. SOC. VOL. 125, NO. 18, 2003

Hydrophobic Base Pairs with Shape Complementarity
A R T I C L E S
Scheme 1 a
a Conditions: (a), NaH, CH3CN, room temperature, 60%; (b), NH3-MeOH, room temperature, 74%; (c), DMTr-Cl, pyridine, room temperature, 99%; (d),
ClP(N-iPr2)(OCH2CH2CN), iPr2EtN, THF, room temperature, quant.; (e), POCl3, proton sponge, (CH3O)3PO, 0 °C, then tri-n-butylamine, bis(tributylam-
monium)pyrophosphate. Abbreviations: Tol, toluoyl; DMTr, dimethoxytrityl; iPr, isopropyl.
Table 1. Steady-State Kinetic Parameters for Insertion of Single Nucleotides into a Template-Primer Duplex by the Exonuclease-Deficient
Klenow Fragmenta
Primer 1, 5′-ACTCACTATAGGGAGGAAGA
Template 1, 3′-TATTATGCTGAGTGATATCCCTCCTTCTNTCTCGA
b
1
1
1
template (N)
nucleoside triphosphate
Km
(
µM)
Vmaxd (%
‚
min-
)
efficiency, Vmax/Km (%‚ ‚ )
min- M-
Q
A
G
C
T
Q
Q
Q
Q
Q
A
G
isoG
isoG
isoG
A
Pa
Pa
Pa
Pa
Pa
A
G
C
T
F
F
F
Pa
C
240 (80)
520 (210)
460 (160)
n.d.c
430 (270)
91 (39)
190 (80)
690 (130)
370 (110)
70 (11)
170 (40)
1800 (1200)
n.d.c
30 (7)
29 (11)
1.3 × 105
5.6 × 104
5.0 × 102
2.8 × 102
1.3 × 104
1.1 × 103
1.1 × 104
8.9 × 103
2.1 × 105
1.4 × 105
2.9 × 102
0.23 (0.06)
n.d.c
0.12 (0.05)
1.2 (0.5)
0.21 (0.06)
7.7 (2.5)
3.3 (0.9)
15 (2)
24 (5)
0.52 (0.27)
n.d.c
390 (120)
340 (100)
1.6 (0.7)
0.93 (0.23)
69 (10)
2.1 (0.7)
2.4 × 103
2.0 × 105
1.3 × 106
T
T
a Assays were performed at 37 °C for 1-20 min using 5 µM template-primer duplex, 5-50 nM enzyme, and 0.6-2100 µM nucleoside triphosphate in
a solution (10 µL) containing 50 mM tris-HCl (pH 7.5), 10 mM MgCl2, 1 mM DTT, and 0.05 mg/mL bovine serum albumin. b Standard deviations are given
in parentheses. c No inserted products were detected after an incubation of 20 min with 1500 or 2100 µM nucleoside triphosphate and 50 nM enzyme.
d Values were normalized to the enzymatic concentration (20 nM) for the various enzyme concentrations used. Standard deviations are given in parentheses.
of Avian myeloblastosis virus (AMV-RT) (Life Sciences Inc., St.
Petersburg, FL) diluted in the annealing buffer. The concentrations used
for primer extension were as follows: primer-template, 150 nM; AMV-
RT, 0.1 unit/µL; and dNTPs, 100 µM each; the incubation time for
each was 20 min. Unit definition: One unit catalyzes the incorporation
of 1 nmol of dTMP into an acid-insoluble product in 10 min at 37 °C
using poly[(rA)‚oligo(dT)] as a template-primer.
NMR Spectra. NMR experiments were performed on a Bruker
model DRX600 spectrometer. Proton assignments were made using
standard two-dimensional (2D) techniques, including NOESY (mixing
times of 75, 150, 225, 300, and 375 ms), DQF-COSY, and NOESY
with jump and return water suppression (mixing time of 150 ms). Data
processing was done using Felix950 software (BIOSYM/Molecular
Simulations). All structural restraints involving nonexchangeable
protons were derived from NOESY data acquired at 15 °C with a
mixing time of 75 ms. Restraints for exchangeable protons were derived
from NOESY with jump and return water suppression data acquired at
3 °C with a mixing time of 150 ms. The backbone conformation and
the sugar pucker were investigated at 15 °C using one-dimensional
(1D) 31P and DQF-COSY experiments, respectively.
two-spin cross-relaxation approximation, with the usual r-6 dependence
on the proton-proton distance. Limits of (30% were placed about
the calculated interproton distances.27 A total of 232 restraints involving
nonexchangeable protons were derived. Sixty exchangeable proton
restraints were given an upper bound of 5 Å and a lower bound of 1.5
Å. Dihedral angle restraints were used to preserve a right-handed DNA
helix.28 Pseudorotational phase angle restraints were derived on the basis
of the H1′-H2′/H2′′ and H3′-H4′ correlations in the COSY spectrum.
A total of 292 NOE distance restraints and 132 dihedral angle restraints
were used for the calculations. Watson-Crick base pairs were identified
using two criteria: the observation of an NH or NH2 proton resonance
significantly downfield-shifted and the observation of strong G-C
NH-NH2 or A-T H2-NH NOEs. In addition, 56 restraints were
included to maintain the Watson-Crick pairing for base pairs 1-5
and 7-12.23,29
Molecular Dynamics. All structural calculations and analyses were
conducted using the AMBER 6.0 program. The calculations used the
AMBER force field in addition to the flat-bottom restraint potentials,
with force constants of 50 kcal‚mol-1‚Å-2 for the distance and 50
(27) Burkard, M. E.; Turner, D. H. Biochemistry 2000, 39, 11748-11762.
(28) Clore, G. M.; Oschkinat, H.; McLaughlin, L. W.; Benseler, F.; Happ, C.
S.; Happ, E.; Gronenborn, A. M. Biochemistry 1988, 27, 4185-4197.
(29) Schmitz, U.; James, T. L. Methods Enzymol. 1995, 261, 3-44.
Restraint Generation. Distance constraints were generated from
the 75-ms-mixing-time NOE volumes with the pyrimidine H5/H6 cross
peaks as a reference at 2.45 Å. All distances were calculated from the
9
J. AM. CHEM. SOC. VOL. 125, NO. 18, 2003 5301

A R T I C L E S
Mitsui et al.
Table 2. Steady-State Kinetic Parameters for Insertion of Single Nucleotides into a Template-Primer Duplex by the Exonuclease-Deficient
Klenow Fragmenta
Primer 2, 5′-ACTCACTATAGGGAGCTTCT
Template 2, 3′-TATTATGCTGAGTGATATCCCTCGAAGANAGAGCT
b
1
1
1
template (N)
nucleoside triphosphate
Km
(µ
M)
Vmaxc (%
‚
min-
)
efficiency, Vmax/Km (%‚ ‚ )
min- M-
Pa
Pa
Pa
Pa
Pa
A
G
C
T
F
F
F
Pa
C
T
isoC
isoC
isoC
isoC
T
Q
A
G
C
T
Q
Q
Q
Q
Q
A
G
isoG
isoG
isoG
isoG
Q
140 (20)
370 (110)
330 (100)
1600 (700)
970 (140)
200 (30)
190 (10)
280 (40)
220 (60)
50 (6)
110 (10)
53 (3)
1.9 (0.4)
0.27 (0.07)
0.83 (0.10)
20 (3)
13 (1)
17 (3)
20 (5)
86 (16)
7.9 × 105
1.4 × 105
5.8 × 103
1.7 × 102
8.6 × 102
1.0 × 105
6.8 × 104
6.1 × 104
9.1 × 104
1.7 × 106
1.2 × 106
3.4 × 102
1.6 × 103
1.1 × 103
1.4 × 105
7.0 × 104
1.8 × 104
1.0 × 104
1.3 × 103
2.8 × 106
76 (3)
91 (28)
530 (240)
1200 (500)
360 (60)
610 (110)
530 (80)
320 (110)
740 (170)
490 (130)
3.1 (1.6)
0.18 (0.06)
1.9 (0.6)
0.41 (0.09)
88 (11)
37 (8)
5.7 (0.5)
7.4 (1.0)
0.66 (0.09)
8.7 (2.6)
A
G
A
a Assays were performed at 37 °C for 1-22 min using 5 µM template-primer duplex, 3-45 nM enzyme, and 0.6-2100 µM nucleoside triphosphate in
a solution (10 µL) containing 50 mM tris‚HCl (pH 7.5), 10 mM MgCl2, 1 mM DTT, and 0.05 mg/mL bovine serum albumin. b Standard deviations are given
in parentheses. c The values were normalized to the enzymatic concentration (20 nM) for the various enzyme concentrations used. Standard deviations are
given in parentheses.
Table 3. Proton Chemical Shifts for the Pa-Q Duplexa
H8/H6
H5/Met/H2
H1
′
H2
′
H2′′
H3
′
H4
′
amino
imino
C1
G2
C3
A4
T5
Pa6
G7
T8
T9
A10
C11
C12
G13
G14
T15
A16
A17
C18
Q19
A20
T21
G22
C23
G24
7.75
8.08
7.51
8.47
7.18
7.60 (H5)
7.93
7.38
7.48
8.43
7.46
7.71
7.99
7.95
7.37
8.37
8.22
6.00
na
na
na
na
na
na
na
na
na
5.86
6.03
5.74
6.36
5.90
6.53
6.06
6.08
5.83
6.30
6.03
6.29
5.80
6.11
5.72
6.04
6.17
5.56
6.16
6.17
5.80
5.94
5.87
6.27
2.10
2.80
2.20
2.81
1.61
2.69
2.69
2.21
2.21
2.83
2.16
2.35
2.69
2.74
2.19
2.86
2.65
1.88
2.78
2.65
2.07
2.71
2.03
2.73
2.53
2.88
2.54
3.01
2.18
2.69
2.86
2.64
2.57
2.97
2.51
2.35
2.81
2.90
2.51
3.01
2.93
2.26
2.93
2.95
2.46
2.77
2.45
2.47
4.82
5.10
4.98
5.13
4.85
5.04
5.04
4.96
4.99
5.14
4.86
4.64
4.92
5.09
4.99
5.18
5.13
4.87
5.12
5.08
4.95
5.08
4.93
4.79
4.18
4.48
4.32
4.54
4.15
4.41
4.50
4.25
4.23
4.54
4.27
4.12
4.29
4.51
4.31
4.53
4.54
4.17
4.46
4.54
4.23
4.46
4.29
4.29
8.12/7.02
na
13.00
na
na
13.32
na
12.03
13.80
13.62
na
na
na
12.76
na
13.44
na
na
na
na
na
13.42
12.59
na
7.74
1.63
5.15
6.06 (H4)
na
1.35
1.75
7.59
5.45
5.77
na
8.31/6.50
7.65/6.37
na
na
na
6.32 (H3)
na
na
na
na
na
na
na
na
na
na
na
8.85 (H6)
na
na
na
na
na
na
na
na
na
na
na
na
na
na
7.76/6.21
8.15/6.67
8.18/6.89
na
1.55
7.09
7.63
5.33
2.06 (Met)
7.71
1.39
na
na
7.61/6.27
7.59/5.94
7.95/6.81
na
7.15/6.17
na
7.29
8.43 (H2)
8.21
7.16
7.96
na
na
5.89 (H8)
na
na
na
na
7.24 (H7)
na
na
na
na
7.46
8.06
5.51
na
8.35/6.59
na
na
na
a Non-exchangeable proton chemical shifts were measured at 15 °C and referenced to the HDO signal at 4.88 ppm, and exchangeable proton chemical
shifts were measured at 3 °C and referenced to the HDO signal at 5.00 ppm. Throughout the table, “na” denotes not applicable.
kcal‚mol-1‚rad-2 for the torsion angle restraints. The starting structure
generation and the visualization of the calculated structures were done
using InsightII 98 software (BIOSYM/Molecular Simulations). The
partial charges for Pa and Q were assigned by ab initio methods at the
RHF/6-31G(d) level, using Gaussian 94 (Gaussian, Inc.). The partial
charges used in the molecular dynamics simulation for the Pa base are
the following: N1, -0.63; C2, 0.20; C3, -0.24; H3, 0.22; C4, -0.30;
H4, 0.21; C5, 0.07; H5, 0.22; C6, 0.33; O6, -0.54; and H6, 0.16. The
partial charges for the Q base are the following: N1, -0.70; C2, 0.31;
H2, 0.22; N3, -0.58; C4, 0.12; C5, 0.63; N6, -0.61; C7, 0.06; H7,
0.20; C8, -0.32; H8, 0.20; C9, 0.09; C10Me, -0.49; and C7MeH,
0.19. Structures were determined by restrained molecular dynamics
simulations and energy minimization. Fifty calculations were performed
using B-form DNA as the starting structure, and a second set of 50
calculations was performed with an A-form starting structure. The two
sets of 50 structures were averaged and subjected to an additional 100
iterations of steepest-descents energy minimization with full restraints
to generate the final structures.
Results and Discussion
Synthesis of the 2′-Deoxyribonucleotide Derivatives of
Pyrrole-2-carbaldehyde (Pa) and Thermal Stability of the
DNA Duplexes Containing Pa. The synthesis of the ribo-
nucleosides of pyrrole-2-carbaldehyde via pyrrole-2-carboxylic
9
5302 J. AM. CHEM. SOC. VOL. 125, NO. 18, 2003

Hydrophobic Base Pairs with Shape Complementarity
A R T I C L E S
acid derivatives was previously reported.30 To facilitate the
synthesis, we directly synthesized the 2′-deoxyribonucleoside
of Pa, by the coupling reaction of pyrrole-2-carbaldehyde with
1-chloro-2-deoxy-3,5-di-O-toluoyl-R-D-erythropentofuranose,
which was obtained as a single product in a 44% yield after
deprotection and purification (Scheme 1). This type of direct
glycosylation, via the sodium salt of pyrrole derivatives with
the R-configuration of the 1-chloro-sugar, reportedly gives 2′-
deoxy-â-nucleosides specifically. The nucleoside of Pa was
1
characterized by H NMR and 13C NMR spectra and by mass
spectrometry, and the production of the 2′-deoxy-â-nucleoside
was confirmed (see the Supporting Information). The pattern
of the anomeric proton signal was identical with that published
for the 2′-deoxy-â-nucleosides of pyrrole derivatives, such as
pyrrole-2-carbonitrile,31 3-nitropyrrole,32 and pyrrolopyrim-
idines.33,34 The 1H NMR spectrum of the 2′-deoxyribonucleoside
of Pa showed the characteristic triplet for H1′ at 6.73 ppm and
the narrow multiplet for H2′ and H2′′, which are patterns similar
to those of other â-isomers of pyrrole derivatives.31-34 Further-
more, NOEs between H1′ and H4′ and between H1′ and H2′′
were observed, suggesting the â-isomer of the product. The
nucleoside of Pa was converted to the amidite and to the
triphosphate by conventional methods. The molar absorption
coefficient of the triphosphate (14 000 at λmax ) 291 nm) was
determined by a quantitative analysis of the phosphorus, after
dephosphorylation of the triphosphate with calf intestine alkaline
phosphatase (TaKaRa, Tokyo).
We first synthesized a trimer d(TPaT) to examine the side
reactions of the Pa-amidite during DNA chemical synthesis.
The coupling efficiency of the amidite of Pa was >98% on a
DNA synthesizer (Applied Biosystems), and the production of
the trimer was confirmed by mass spectrometry (see the
Supporting Information). After synthesis and deprotection, the
trimer was analyzed by HPLC, and no byproduct was observed
after treatments with 3.5% dichloroacetic acid in dichlo-
romethane at room temperature for 1 h, followed by concen-
trated ammonia at 60 °C for 7 h, or with 0.02 M iodine in a
solution containing 20% pyridine, 70% THF, and 10% water
at room temperature for 1 h, followed by concentrated ammonia
at 60 °C for 7 h. The DNA fragments (12-mer and 35-mer)
containing Pa were then synthesized.
The Q-Pa pair in the DNA duplex showed selective thermal
stability. The stability of the DNA duplex (5 mM) of 5′-
GGTAACQATGCG and 5′-CGCATPaGTTACC (Tm ) 39.6
°C) was higher than that of a duplex containing an A-Pa pair
(Tm ) 34.2 °C) or a G-Pa pair (Tm ) 36.0 °C), although the
Q-Pa duplex was less stable than the natural A-T duplex (Tm
) 49.7 °C). The thermal stability of the Q-Pa duplex was
compared to that of the Q-F, A-F, and G-F duplexes. The
Tm values of duplexes containing each of the Q-F, A-F, and
G-F pairs are 41.1, 37.6, and 37.6 °C, respectively. The lower
stability of the Q-Pa duplex, as compared to the Q-F duplex,
may be because the stacking interaction of a five-membered
Figure 2. Primer extensions using natural and unnatural nucleotides in
the templates and as substrates. (A) Sequences of the 23-mer primer and
the 35-mer templates, templates 1 and 2, used in the reactions. (B and C)
Autoradiograms of a denaturing polyacrylamide gel, showing primer
extensions with the 3′ f 5′ exonuclease-proficient Klenow fragment [KF
(exo+)]; data were obtained at 37 °C, using 0.1 unit/µL enzyme, 200 nM
primer-template duplex, 10 µM dNTP, and a reaction time of 5 min. (D)
Autoradiogram of a denaturing polyacrylamide gel, showing primer
extensions with Avian myeloblastosis virus reverse transcriptase (AMV-
RT); data were obtained at 37 °C using 0.1 unit/µL enzyme, 150 nM
primer-template duplex, 100 µM dNTP, and a reaction time of 20 min.
(30) Meade, E. A.; Wotring, L. L.; Drach, J. C.; Townsend, L. B. J. Med. Chem.
1992, 35, 526-533.
(31) Ramasamy, K.; Robins, R. K.; Revankar, G. R. Tetrahedron 1986, 42,
5869-5878.
(32) Bergstrom, D. E.; Zhang, P.; Toma, P. H.; Andrews, P. C.; Nichols, R. J.
Am. Chem. Soc. 1995, 117, 1201-1209.
(33) Kazimierczuk, Z.; Cottam, H. B.; Revankar, G. R.; Robins, R. K. J. Am.
Chem. Soc. 1984, 106, 6379-6382.
ring, such as Pa, with neighboring bases is weaker than that of
a six-membered ring, such as that observed in natural pyrimidine
(34) Robins, M. J.; Robins, R. K. J. Am. Chem. Soc. 1965, 87, 4934-4940.
9
J. AM. CHEM. SOC. VOL. 125, NO. 18, 2003 5303

A R T I C L E S
Mitsui et al.
Figure 3. Base-to-H1′ region of a 75-ms NOESY spectrum obtained at 15 °C.
bases.35 This instability is also present in the other base pairs
with five-membered rings. For example, a DNA duplex (13-
mer) containing an unnatural self-pair between 2-methyl-
thiophenes (Tm ) 52.1 °C) is less stable than that containing
the A-T pair (Tm ) 59.2 °C).32 Interestingly, relative to the
A-F duplex, the A-Pa duplex was destabilized by 3.4 °C,
showing the poor fitting between A and Pa, in comparison to
that between A and F.
was efficiently inserted into DNA opposite Q in the template.
The incorporation efficiency of Pa into DNA opposite Q (Vmax
/
KM ) 1.3 × 105 %‚min-1‚M-1) was higher than that of T
opposite Q (Vmax/KM ) 8.9 × 103 %‚min-1‚M-1) as well as
that of the previously developed s-y pair (the incorporation of
y opposite s; Vmax/KM ) 5.9 × 104 %‚min-1‚M-1).
We compared the incorporation efficiency and selectivity of
the Q-Pa pairing with the Q-F pairing (Tables 1 and 2). The
T analogue, F, efficiently paired with both Q and A. For
example, the incorporation efficiencies of F opposite Q and A
were Vmax/KM ) 2.1 × 105 and 1.4 × 105 %‚min-1‚M-1, respec-
tively. In contrast, the Q-Pa pairings in the insertion experi-
ments, especially the incorporation of Q opposite Pa (Vmax/KM
) 7.9 × 105 %‚min-1‚M-1), were more efficient than the
Single-Nucleotide Insertion Experiments. The efficiency
and selectivity of the Q-Pa pairing in replication were assessed
by the steady-state kinetics of single-nucleotide insertion
experiments.25,26 The exonuclease-deficient Klenow fragment
was used with combinations of a nucleoside triphosphate and a
partially double-stranded template (35-mer) with a 32P-labeled
primer (20-mer), in which various bases in the template were
adjacent to the 3′ end of the primer. The insertion of each
substrate opposite each base in the template was analyzed by
gel electrophoresis, and the kinetic parameters of each base
pairing were determined (Tables 1 and 2). The substrate of Pa
noncognate pairing of the substrate A opposite Pa (Vmax/KM
)
1.4 × 105 %‚min-1‚M-1). This suggests that the fitting of the
five-membered-ring base Pa with the hydrophilic natural purine
bases is inferior, in compairson to that of the six-membered-
ring bases, such as F and the natural pyrimidines, C and T.
The Pa-Q pairing in replication showed the highest selectiv-
ity, relative to the noncognate pairings with the natural bases.
(35) Kool, E. T. Annu. ReV. Biophys. Biomol. Struct. 2001, 30, 1-22.
9
5304 J. AM. CHEM. SOC. VOL. 125, NO. 18, 2003

Hydrophobic Base Pairs with Shape Complementarity
A R T I C L E S
Table 4. Structural Statistics for the Pa-Q Duplex
Number of Restraints
total
480
176
116
60
distance restraints
intraresidue
interresidue
exchangeable
non-exchangeable
hydrogen bonding
232
56
132
dihedral angle restraints
Violations of Experimental Restraints in the Final Structure When:a
b
A-form DNA was used as the starting structure
distance violations (0.0.1 Å)
total
intraresidue
interresidue
SA
0
0
0
0
dihedral violations (>2°)
distance violations (0.0.1 Å)
total
b
B-form DNA was used as the starting structure
SA
0
0
0
0
intraresidue
interresidue
dihedral violations (>2°)
Atomic rms Differences (Å)c
final(A-form starting) and final(B-form starting)
final(A-form starting) and final(B-form starting) (residues 6 and 19)
final(B-form starting) and B-form starting structure
2.44
0.18
4.23
a Not including the residues of the 3′- or 5′-terminus (residues 1, 12, 13, and 24). b Energy-minimized average structure from 50 calculations. c Average
pairwise rms differences were calculated using energy-minimized average structures.
The efficiency of Pa pairing opposite Q was 10-118 times
higher than those of the natural bases opposite Q. Similarly,
the incorporation efficiency of Q opposite Pa was 6-4600 times
higher than those of the natural bases opposite Pa. In contrast,
the incorporation efficiencies of Pa or Q opposite the natural
bases were lower (>23 times) than those of both T opposite A
and A opposite T. In this context, the incorporation of Pa
opposite A was somewhat high (Vmax/KM ) 5.6 × 104
%‚min-1‚M-1), but the efficiency was much lower than that of
the incorporation of T opposite A (Vmax/KM ) 1.3 × 106
%‚min-1‚M-1), suggesting that T might be predominantly
incorporated opposite A. Thus, the Pa-Q pairing exhibited
specificity and may work with the natural base pairs in
replication.
To determine the utility of the Q-Pa pair with a combination
of other unnatural base pairs as the third and fourth pairs, we
performed the cross comparisons of the incorporation selectivity
between the Q-Pa and the hydrophilic isoG-isoC pairs. The
incorporation by all combinations among Q, Pa, isoG, isoC
(5-methylisocytosine), and natural bases as substrates and
template bases was conducted using the Klenow fragment
(Tables 1 and 2), except for the incorporation of d(isoC)TP,
because of the chemical instability in the assay system. The
isoG-Pa pairing was inferior to the Q-Pa and isoG-isoC
pairings, and in particular, the incorporation of Pa (ranging in
concentration from 0.06 mM to 1.5 mM) opposite isoG was
not observed. Although the incorporation of Q opposite isoC
showed relatively high efficiency (Vmax/KM ) 1.8 × 104
%‚min-1‚M-1), the incorporation was less efficient than that
of isoG opposite isoC (Vmax/KM ) 7.0 × 104 %‚min-1‚M-1).
This high efficiency of the Q-isoC pairing may be due to the
favorable shape complementarity between Q and isoC. Simi-
Primer Extension. Next, we examined the extension after
the incorporation of Pa or Q into the primers by the exonu-
clease-proficient (exo+) Klenow fragment and AMV-RT. The
primer extension reactions with combinations of the several
substrates and templates were conducted using the duplex DNA
between a 23-mer primer and 35-mer templates, template 1 for
the incorporation of Pa, and template 2 for the incorporation
of Q (Figure 2A). The extension was analyzed by gel electro-
phoresis, as shown in Figure 2B-D. The full-extension products
were observed as 33-mer fragments, because of the absence of
a substrate (dCTP for template 1 and dGTP for template 2)
opposite the bases at position 34 in the templates.
The extension including the Q-Pa pairing at position 28 by
the Klenow fragment (exo+) was observed with high efficiency
and selectivity (Figure 2B). There were no terminated products
after the incorporations of Pa opposite Q and of Q opposite
Pa, and the 33-mer products were obtained (Figure 2B, lanes 7
and 13). In contrast, the extension after the incorporation of Pa
opposite A was much less efficient than that of the T-A and
Pa-Q pairings (see Figure 2B, lanes 3, 4, 7, and 13), and the
extensions involving the incorporations of T opposite Q, Q
opposite T, and A opposite Pa were paused at positions 28 and/
or 29 (see Figure 2B, lanes 6, 10, and 12).
Interestingly, the extension involving the Q-F pairing by
the Klenow fragment (exo+) was much less effective (Figure
2C). The substrate of F was efficiently incorporated opposite
Q at position 28 in the template, but the extension after the
incorporation did not proceed (Figure 2C, lanes 4 and 7). It is
known that the minor groove interaction between F in the primer
and the Klenow fragment is too small to process the subsequent
extension effectively, although the Q-F pair is relatively stable
against the exonuclease activity of the Klenow fragment.7,21,22
Thus, the Q-Pa pair has more efficient processivity than that
of the Q-F pair.
larly, the incorporation of isoG opposite T was very high (Vmax
/
KM ) 1.4 × 105 %‚min-1‚M-1), but this incorporation was less
effective than that of A opposite T (Vmax/KM ) 2.8 × 106
%‚min-1‚M-1). Thus, for practical use, the selective incorpora-
tion of isoG into DNA opposite isoC can be combined with
the Q-Pa pairing.
The substrate of F was not recognized by AMV-RT and was
not incorporated into DNA.21 In contrast, the substrate of Pa
was incorporated into DNA opposite Q by AMV-RT, and the
extension proceeded after the incorporation of Pa into the primer
9
J. AM. CHEM. SOC. VOL. 125, NO. 18, 2003 5305

A R T I C L E S
Mitsui et al.
Figure 4. Three-dimensional structures of the Q-Pa duplex. Depiction A shows the superimposition of 10 randomly selected final structures after restrained
molecular dynamics and energy minimization; five started from B-form DNA, whereas the other five started from A-form DNA. Depiction B shows the
superimposition of the Pa-Q pairs in the 10 structures shown in depiction A. Depiction C shows a stereo view from the major groove of the central three
base pairs for the Pa-Q duplex, showing the Pa6 (green) and Q19 (yellow) pair, as well as their immediate neighbors (blue). Depiction D shows a cutaway
view along the helical axis.
(Figure 2D, lane 7). Similarly, the extension proceeded after
the incorporation of Q opposite Pa (Figure 2D, lane 13). These
results of the extensions by AMV-RT, as well as the Klenow
fragment (exo+), show that the aldehyde group of Pa was well
recognized by the DNA polymerases, in comparison with the
fluoro group of F.
Structural Analysis. To assess the steric fitting of the bases
in the duplex, we determined the geometry of the Q-Pa pair
in a DNA duplex by NMR. Structural studies were performed
at 600 MHz in an aqueous solution using the Q-Pa contain-
ing duplex, 5′-C1G2C3A4T5Pa6G7T8T9A10C11C12 and 5′-
G13G14T15A16A17C18Q19A20T21G22C23G24. The same
sequences of the Z-F duplex used for the structural analysis
were employed to compare the base-pair fitting of the Q-Pa
pair with the Z-F pair.23 Nonexchangeable proton signals were
assigned by standard two-dimensional techniques, including
DQF-COSY and NOESY (Table 3). The chemical shifts of
the imino protons for the G-C and A-T base pairs were
consistent with the formation of Watson-Crick base pairs. The
cross peaks in a NOESY spectrum recorded in an H2O solution
also confirmed the formation of Watson-Crick base pairs in
the duplex. The NOE connectivities of the base to H1′ (Figure
3) to H2′/2′′, and to H3′, were observed throughout both strands,
showing the continuous stacking between the bases in the
duplex. The observed base to H1′ intensities indicated that all
the bases adopted an anti conformation. In addition, the
resolvable DQF-COSY cross-peak patterns indicated an S (C2′-
endo) sugar pucker. The 31P NMR spectrum showed that all
9
5306 J. AM. CHEM. SOC. VOL. 125, NO. 18, 2003

Hydrophobic Base Pairs with Shape Complementarity
A R T I C L E S
fluctuations of each structure are relatively large, especially at
both termini, and are larger than those of the related Z-F duplex
obtained by NMR.23 These may be due to the weak stacking
stability of Pa, which renders the structural flexibility in a
solution.
The cutaway view along the helical axis shows that the actual
geometry of the Q-Pa pair (Figure 4B and D) is similar to our
expected structure, as shown in structure D in Figure 1. The
conformation of the Q-Pa pair closely resembles that of the
Watson-Crick base pairs (Figure 5). The oxygen of the
aldehyde group of Pa is located in a slightly different place
than in the 2-keto group of T. The polymerases, such as the
Klenow fragment and AMV-RT, must tolerate this difference
for the minor groove interaction, or the flexibility of the Pa
base may be favorable for the recognition.
Figure 5. Superimposition of the Q-Pa pair (green-yellow) on the A-T
pair (blue-red) in the canonical B-form conformation.
the phosphorus resonances resided within a 1 ppm range for
the duplex, which is consistent with a B-form DNA structure.
Several NOEs were observed among the Q-Pa pair and the
neighboring bases. The A20(H2) and T5(H6) protons exhibit
NOEs to the Pa6(H6) and Pa6(H4) protons, respectively. In
addition, the T5(H3) proton exhibits NOEs to the Pa6(H6,H4),
and Q19(H8) protons, and the G7(H1) proton exhibits NOEs
to the Pa6(H6,H3) and Q19(H7,H8) protons. Similarly, NOEs
between Q19(H7) and Pa6(H6,H3), between Q19(H8) and Pa6-
(H6, H3), and between Q19(Me) and Pa6(H6,H3,H4) were
observed. These NOE connectivities are consistent with a
predominant conformation, in which both Pa6 and Q19 are
stacked within the DNA helix.
The structural determination of the duplex containing the
Q-Pa pair was started from both A-form and B-form 12-mer
duplexes; a series of simulated annealing calculations was
performed using the AMBER force field with 292 distance
restraints and 132 dihedral-angle restraints (Table 4). The
convergence of the structures is illustrated in Figure 4A and B,
and the stereoviews of the central three base pairs for the Q-Pa
duplex that were averaged and subjected from the 100 structures
are shown in Figure 4C and D. The duplex adopts the B-form
structure. The sugar puckers of Pa6 and Q19 are both of the
S-type, and both of the bases adopt anti glycosidic orientations,
stacking with neighboring bases. The duplex seems to bend as
an entire unit, and both the Q19 and Pa6 bases are slightly tilted
to each opposite direction. However, the NMR signals derived
from the Q-Pa pair are relatively broad, suggesting that the
base pair is flexible in solution. As shown in Figure 4A, the
Conclusion
As a substrate and a base in templates, the hydrophobic base
combined with the aldehyde group, and the five-membered ring,
can be recognized by DNA polymerases and function in
replication. In addition, the unique specificity of each of the
Q-Pa and Q-F pairings in replication (where Q is 9-meth-
ylimidazo[(4,5)-b]pyridine, Pa is pyrrole-2-carbaldehyde, and
F is 2,4-difluorotoluene) shows that the selectivity of an
unnatural base pair can be increased by fine-tuning the shape
complementarity. In contrast to the specific Q-F and A-F
pairing, our biological and structural studies show that the Q-Pa
pairing is favored over the A-Pa pairing, because of the better
shape-fitting between Q and Pa and the weak stacking ability
of Pa, which destabilizes the pairing with the water-solvated
A. The framework of the Pa base, as well as that of other five-
membered-ring bases,36-38 would be useful for the further
development of unnatural base pairs, and studies of practical
uses of the Q-Pa pairing are in progress.
Supporting Information Available: NMR, NOE, and MS
data for the characterization of the nucleoside of Pa; MS data
of the trimer of d(TPaT) (PDF). This material is available free
of charge via the Internet at http://pubs.acs.org.
JA028806H
(36) Berger, M.; Luzzi, S. D.; Henry, A. A.; Romesberg, F. E. J. Am. Chem.
Soc. 2002, 124, 1222-1226.
(37) Hirao, I.; Mitsui, T.; Fujiwara, T.; Kimoto, M.; To, T.; Okuni, T.; Sato,
A.; Harada, Y.; Yokoyama, S. Nucleic Acids Res. Suppl. 2001, 1, 17-18.
(38) Jiang, X.-J.; Kalman, T. I. Nucleosides Nucleotides 1994, 13, 379-388.
9
J. AM. CHEM. SOC. VOL. 125, NO. 18, 2003 5307