An unnatural hydrophobic base, 4-propynylpyrrole-2-carbaldehyde, as an efficient pairing partner of 9-methylimidazo[(4,5)-b]pyridine

Article abstract of DOI:10.1016/j.bmcl.2003.09.059

To develop unnatural base pairs that function in replication, we designed 4-propynylpyrrole-2-carbaldehyde (designated as Pa′) and synthesized the nucleoside derivatives of Pa′. The base pairing of Pa′ with the partner, 9-methylimidazo[(4,5)-b]pyridine (Q), was compared to that of pyrrole-2-carbaldehyde (Pa), which was previously developed as a specific pairing partner of Q. The thermal stability of a DNA duplex containing the Q-Pa′ pair and the incorporation efficiency of the Pa′ substrate (dPa′TP) into DNA opposite Q by the Klenow fragment of Escherichia coli DNA polymerase I were improved, in comparison with those of the Q-Pa pair. These improvements result from the increased hydrophobicity and stacking stability of Pa′ by the introduction of the propynyl group to Pa, providing valuable information for the further development of unnatural base pairs toward the expansion of the genetic alphabet.

Full text of DOI:10.1016/j.bmcl.2003.09.059

Bioorganic & MedicinalChemistry Letters 13 (2003) 4515–4518
An Unnatural Hydrophobic Base,
4-Propynylpyrrole-2-carbaldehyde, as an Efficient Pairing Partner
of 9-Methylimidazo[(4,5)-b]pyridine
Tsuneo Mitsui,a Michiko Kimoto,b Akira Sato,b Shigeyuki Yokoyamab,c,d,
and Ichiro Hiraoa,b,
*
*
aResearch Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Komaba, Meguro-ku,
Tokyo 153-8904, Japan
bProtein Synthesis Technology Team, Protein Research Group, RIKEN Genomic Sciences Center (GSC),
1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan
cDepartment of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo,
Bunkyo-ku, Tokyo 113-0033, Japan
dRIKEN Harima Institute at Spring-8, 1-1-1 Kouto, Mikazuki-cho, Sayo, Hyogo 679-5148, Japan
Received 7 July 2003; accepted 31 July 2003
Abstract—To develop unnatural base pairs that function in replication, we designed 4-propynylpyrrole-2-carbaldehyde (designated as
Pa0) and synthesized the nucleoside derivatives of Pa0. The base pairing of Pa0 with the partner, 9-methylimidazo[(4,5)-b]pyridine (Q),
was compared to that of pyrrole-2-carbaldehyde (Pa), which was previously developed as a specific pairing partner of Q. The thermal
stability of a DNA duplex containing the Q–Pa0 pair and the incorporation efficiency of the Pa0 substrate (dPa0TP) into DNA opposite
Q by the Klenow fragment of Escherichia coli DNA polymerase I were improved, in comparison with those of the Q–Pa pair. These
improvements result from the increased hydrophobicity and stacking stability of Pa0 by the introduction of the propynylgroup to Pa,
providing valuable information for the further development of unnatural base pairs toward the expansion of the genetic alphabet.
# 2003 Elsevier Ltd. All rights reserved.
The creation of unnaturalbase pairs that can work with
the naturalA–T and G–C base pairs in the celexpands
the genetic alphabet, leading to novel biotechnology. In
addition, the unnaturalbase pairs are usefulfor under-
standing the mechanisms of replication, transcription, and
translation. Recent advancements in the development of
unnaturalbase pairs have been achieved by the use of
hydrophilic1ꢀ4 and hydrophobic5ꢀ8 base analogues. One
of the hydrophobic base pairs, 9-methylimidazo[(4,5)-
b]pyridine (Q) and 2,4-difluorotoluene (F), was developed
as the isostere of the A–T pair, and Q and F can work in
replication as A and T analogues, respectively6 (Fig. 1a
and 1b). In particular, the A–F pair functions as well as
the A–T and Q–F pairs. Thus, the Q–F pair cannot be
used as the third base pair for the expansion of the
genetic alphabet. However, the hydrophobic base pair
revealed the importance of the shape fitting between
pairing bases in replication.9
To confer exclusive selectivity to the Q–F pair, we
developed 4-propynylpyrrole-2-carbaldehyde (Pa0), to
replace F (Fig. 1c). Previously, we developed pyrrole-2-
carbaldehyde (Pa) as a pairing partner of Q10 (Fig. 1d).
The five-membered ring of Pa fits well with Q, but not
with A, and the recognition of the aldehyde group of Pa
by polymerases is superior to that of the fluoro group of
F. However, the stacking stability and the hydro-
phobicity of Pa are inferior to those of F, such that a
DNA duplex containing the Q–Pa pair is less stable
than that containing the Q–F pair, and the enzymatic
incorporation of dPaTP into DNA opposite Q is less
effective than that of dFTP opposite Q.10 To address
these shortcomings, we designed the unnaturalbase,
Pa0, in which the propynylgroup was introduced at
position 4 of Pa. Here, we report the synthesis of the
nucleoside derivatives of Pa0, the thermalstabilty of a
DNA duplex containing the Q–Pa0 pair, and the enzy-
matic incorporation of dPa0TP into DNA.
*Corresponding authors. Tel.: +81-45-503-9644; fax: +81-45-503-
9645; e-mail: ihirao@postman.riken.go.jp (I. Hirao); tel.: +81-3-5841-
4413; fax: +81-3-5841-8057; e-mail: yokoyama@biochem.s.u-tokyo.
ac.jp (S. Yokoyama).
0960-894X/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2003.09.059

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T. Mitsui et al. / Bioorg. Med. Chem. Lett. 13 (2003) 4515–4518
0
Figure 1. Base-pair structures: the naturalA–T pair (a) and the unnaturalQ–F (b), Q–Pa (c), and Q–Pa (d) pairs. R denotes a ribose moiety.
The nucleoside derivatives of Pa0 (4) were synthesized as
shown in Scheme 1. We first synthesized 4-iodopyrrole-
2-carbaldehyde (2)11 to introduce the propynylgroup at
position 4 of pyrrole-2-carbaldehyde (1). During the
process, the aldehyde group was protected with an imi-
nium salt12 for the efficient meta-directed electrophilic
substitution of 1. Then, the iminium salt was converted
to 2 using N-iodosuccinimide, followed by a treatment
with NaHCO3 (57%: two-step totalyiedl ). 12,13 Compound
2 was converted to 4-propynylpyrrole-2-carbaldehyde
(3) by the palladium-mediated coupling with tributyl(1-
propynyl)tin (90%).13 By the same procedure used for
the synthesis of Pa,10 the glycosidation of Pa0 was carried
out using 1-chloro-2-deoxy-3,5-di-O-toluoyl-a-d-erythro-
pentofuranose and 3, activated with NaH, followed by
deprotection of the toluoyl groups with methanolic
ammonia to give 4 with a 36% yield. The nucleoside of
Pa0 (4)14 was characterized by 1H and 13C NMR spectra
and mass spectrometry. The b-configuration of 4 was
confirmed by the NOE conectivities between H10 and
H40 and between H10 and H200 (data not shown). The
nucleoside of Pa0 (4) was converted to the phosphor-
amidite (5) for the chemicalsynthesis of DNA frag-
ments, and to the nucleoside triphosphate (6, dPa0TP)
to function as a substrate in replication. DNA fragments
were synthesized using a DNA synthesizer (PE Applied
Biosystems), and the coupling efficiency of 5 was more
than 98%.
Assessments of the thermalstabiilties of DNA dupelxes
containing Pa0, Pa, F, or Q, were performed using the
DNA fragments, 50-CGCATN1GTTACC (N1=Pa0, Pa,
0
F, or the naturalbases) and 5 -GGTAACN2ATGCG
(N2=Q or the naturalbases), (each 5 mM) in 10 mM
sodium phosphate (pH 7.0) containing 100 mM NaCl
and 0.1 mM EDTA. The stability of the DNA duplex
containing the Q–Pa0 pair (Tm=42.2 ꢁC) was increased,
in comparison to those containing the Q–Pa pair
Scheme 1. Conditions: (a) pyrrolidinium perchlorate, pyrrolidine in benzene, 92%; (b) N-iodosuccinimide in acetonitrile, then NaHCO3, 62%; (c) tri-
butyl(1-propynyl)tin, Pd(PPh3)4, CuI, in CH3CN, 90%; (d) NaH, 1-chloro-2-deoxy3,5-di-O-toluoyl-a-d-erythropentofuranose in CH3CN; (e) NH3 in
MeOH, 36% (two-step yield); (f) DMT-Cl in pyridine; (g) CIP(N-iPr2)(OCH2CH2CN), iPr2EtN in THF, 82% (two-step yield); (h) POCl3, proton sponge
0
in PO(OCH3)3 then tri-n-butylamine, bis(tributylammonium)pyrophosphate in DMF. Abbreviations: DMT, 4,4 -dimethoxytrityl; iPr, isopropyl.

T. Mitsui et al. / Bioorg. Med. Chem. Lett. 13 (2003) 4515–4518
4517
Table 1. Steady-state kinetic parameters for insertion of single nucleotides into a template-primer duplex by the exonuclease-deficient Klenow
fragmenta
Primer
Template
50-ACTCACTATAGGGAGGAAGA
30-TATTATGCTGAGTGATATCCCTCCTTCTNTCTCGA
Template
(N)
ꢀ
Nucleside triphosphate
KM
(mM)
Vmax
(% minꢀ1 d
Efficiency, Vmax/KM
(% minꢀ1 Mꢀ1
)
)
Q
A
G
C
T
Qe
Ae
Ge
Ce
Te
Q
Pa0
Pa0
Pa0
Pa0
Pa0
Pa
Pa
Pa
Pa
Pa
Q
97 (44)b
130 (40)
67 (31)
110 (30)
180 (130)
240 (80)
520 (210)
460 (160)
n.d.c
33 (11)
22 (4)
0.27 (0.11)
0.20 (0.05)
0.37 (0.06)
30 (7)
29 (11)
0.23 (0.06)
n.d.c
0.12 (0.05)
8.2 (1.2)
2.1 (1.3)
3.4ꢂ105
1.7ꢂ105
4.0ꢂ103
1.8ꢂ103
2.1ꢂ103
1.3ꢂ105
5.6ꢂ104
5.0ꢂ102
2.8ꢂ102
1.1ꢂ105
1.3ꢂ106
430 (270)
74 (30)
1.6 (0.7)
A
T
aAssays were carried out at 37 ꢁC for 1–20 min using 5 mM template-primer duplex, 10–50 nM enzyme, and 0.6–2100 mM nucleoside triphosphate in
a solution (10 mL) containing 50 mM Tris–HCl(pH 7.5), 10 mM MgCl , 1 mM DTT, and 0.05 mg/mL bovine serum albumin.
2
bStandard deviations are given in parentheses.
cNo inserted products were detected after an incubation for 20 min with 1500 or 2100 mM nucleoside triphosphate and 50 nM enzyme.
dThe values were normalized to the enzymatic concentration (20 nM) for the various enzyme concentrations used.
eData taken from ref 10.
(Tm=39.6 ꢁC) or the Q–F pair (Tm=41.1 ꢁC). Interest-
ingly, the selectivity of the Q–Pa0 pair was also
improved, relative to the non-cognate A–Pa0 and G–Pa0
pairs; the stability of the Q–Pa0 duplex (Tm=42.2 ꢁC)
was higher than those of the A–Pa0 (Tm=35.2 ꢁC) and
G–Pa0 (Tm=36.2 ꢁC) duplexes. The stability of Pa0
pairing with A or G was not remarkably increased in
comparison to that of Pa pairing with A or G; the Tm
values of the Q–Pa, A–Pa, and G–Pa duplexes were
39.6, 34.2, and, 36.0 ꢁC, respectively.10
relatively small. These results suggest that the hydro-
phobicity and stacking stability of the base affect the
affinity of the substrate for the open complex of the
Klenow fragment, providing insights into the further
development of the unnatural base pairs. Application
and further improvements of the Q–Pa0 pair are being
investigated.
References and Notes
The incorporation efficiency of dPa0TP into DNA was
assessed by single-nucleotide insertion experiments,
using the exonuclease-deficient Klenow fragment15,16
(Table 1). Similar to the thermal stability of Pa0, the
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Hirao, I.; Yokoyama, S. J. Am. Chem. Soc. 2003, 125, 5298.
11. 4-Iodopyrrole-2-carbaldehyde (2): 1H NMR (270 MHz,
DMSO-d6): d 7.12 (m, 1H), 7.34 (m, 1H), 9.42 (d, 1H, J=0.8
Hz), 12.40 (bs, 1H); HRMS (FAB, 3-NBA matrix) calcd for
C5H5NOI (M+1) 221.9416, found 221.9428; 4-Propy-
nylpyrrole-2-carbaldehyde (3): 1H NMR (270 MHz, CDCl3):
d 2.00 (s, 3H), 6.94 (m, 1H), 7.16 (m, 1H), 9.46 (d, 1H, J=1.1
Hz); HRMS (FAB, 3-NBA matrix) calcd for C8H8NO (M+1)
134.0606, found 134.0617.
incorporation efficiency of dPa0TP opposite Q (Vmax
/
KM=3.4ꢂ105% minꢀ1 Mꢀ1) was increased, in comparison
to that of dPaTP opposite Q (Vmax/KM=1.3ꢂ105) or
dFTP opposite Q (Vmax/KM=2.1ꢂ105). Although the
incorporation efficiencies of dPa0TP opposite the natural
bases were also increased, the Q–Pa0 pairing was still
more efficient than the Pa0 pairing with the naturalA, G,
C, and T bases (Vmax/KM=1.7ꢂ105, 4.0ꢂ103, 1.8ꢂ103,
and 2.1ꢂ103, respectively). On the other hand, dQTP was
incorporated into DNA in a self-complementary manner
by the Klenow fragment, and the incorporation efficiency
of dQTP opposite Q (Vmax/KM=1.1ꢂ105) was as high
as that of dPaTP opposite Q. However, this Q–Q pair-
ing was inferior to the Q–Pa0 pairing.
By the introduction of the propynylgroup to Pa, the Q–
Pa0 pair was endowed with efficiency and specificity in
replication. The increase in the incorporation efficiencies
of dPa0TP by the Klenow fragment results from the
decrease in the KM values (67–180 mM), in comparison
to those of dPaTP (240–520 mM, except for opposite C)
(Table 1). In contrast, the difference of the Vmax values
12. Sonnet, P. E. J. Org. Chem. 1971, 36, 1005.
13. Alvarez, A.; Guzman, A.; Ruiz, A.; Velarde, E.;
Muchowski, J. M. J. Org. Chem. 1992, 57, 1653.
between the incorporations of dPa0TP (0.20–33% minꢀ1
and dPaTP (0.13–31% minꢀ1, except for opposite C) is
)

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T. Mitsui et al. / Bioorg. Med. Chem. Lett. 13 (2003) 4515–4518
14. 1-(20-Deoxy-b-d-ribofuranosyl)-4-propynylpyrrole-2-car-
baldehyde (4): 1H NMR (270 MHz, DMSO-d6): d 1.97 (s, 3H),
2.10 (m, 1H), 2.27 (m, 1H), 3.55 (m, 2H), 3.80 (m, 1H), 4.23
(m, 1H), 4.99 (t, 1H, J=5.5 Hz), 5.22 (d, 1H, J=4.0 Hz), 6.65
(t, 1H, J=6.3 Hz), 7.09 (d, 1H, J=1.8 Hz), 7.86 (s, 1H), 9.45
(d, 1H, J=1.0 Hz); 13C NMR (67 MHz, DMSO-d6): d 3.90,
41.95, 61.07, 69.80, 73.21, 85.11, 86.21, 87.53, 105.93, 126.52,
129.99, 130.28, 179.44: HRMS (FAB, 3-NBA matrix) calcd
for C13H16NO4 (M+1) 250.1079, found 250.1040; UV–
vis (in EtOH): lmax 311 nm (7.12ꢂ103), lmax 259 nm
(9.03ꢂ103), lmax 228 nm (15.63ꢂ103), lmin 282 nm (3.50ꢂ103),
lmin 248 nm (7.03ꢂ103); TLC: Rf=0.21 (CH2Cl2/
MeOH=20:1, v/v).
15. 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.
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