Efforts toward expansion of the genetic alphabet: Optimization of interbase hydrophobic interactions

Article abstract of DOI:10.1021/ja0009931

Six novel unnatural nucleobases have been characterized that form stable base pairs in duplex DNA, relying not on hydrogen bonds, but rather on interbase hydrophobic interactions. These nucleobases are derivatives of the hydrophobic base pair between 7-az

Full text of DOI:10.1021/ja0009931

VOLUME 122, NUMBER 32
AUGUST 16, 2000
© Copyright 2000 by the
American Chemical Society
Efforts toward Expansion of the Genetic Alphabet: Optimization of
Interbase Hydrophobic Interactions
Yiqin Wu, Anthony K. Ogawa, Markus Berger, Dustin L. McMinn, Peter G. Schultz,* and
Floyd E. Romesberg*
Contribution from the Department of Chemistry, The Scripps Research Institute,
10550 North Torrey Pines Road, La Jolla, California 92037
ReceiVed March 20, 2000
Abstract: Six novel unnatural nucleobases have been characterized that form stable base pairs in duplex DNA,
relying not on hydrogen bonds, but rather on interbase hydrophobic interactions. These nucleobases are
derivatives of the hydrophobic base pair between 7-azaindole (7AI) and isocarbostyril (ICS). Derivatives of
7AI and ICS were examined that have increased hydrophobic surface area, as well as increased polarizability.
As observed with 7AI and ICS, these derivatives are recognized as substrates by Klenow fragment of Escherichia
coli DNA polymerase I. The unnatural base pair between pyrrolopyrizine (PP) and C3-methylisocarbostyril
(MICS) is enzymatically incorporated into DNA with high efficiency (kcat/KM ) 106 M-1 min-1) and moderate
selectivity. These studies represent a significant step toward the generation of a stable, orthogonal base pair
that can be enzymatically incorporated into DNA with good fidelity.
1. Introduction
κ:X and diso-C:diso-G have been studied extensively. However,
an approach based solely on unique H-bonding patterns limits
the available unnatural nucleobases to fewer than those that can
be constructed by synthetic chemists. Furthermore, this approach
may be limited by the existence of relatively stable base
tautomers that could mispair with native bases in duplex DNA
or during polymerase-catalyzed DNA synthesis.12-15 Consistent
with this notion, it has not been possible to identify a DNA
The structure of duplex DNA is based on the complementary
Watson-Crick hydrogen-bonding patterns of adenine with
thymine (dA:dT base pair) and guanine with cytosine (dG:dC
base pair).1 Previous efforts to expand the number of bases
available for information storage in DNA have used N- or
C-glycosidic nucleosides with purine- or pyrimidine-like nucleo-
bases that have unique patterns of hydrogen bond donors and
acceptors.2-11 For example, the hydrogen-bonding base pairs
(6) Kovacs, T.; Otvos, L. Tetrahedron Lett. 1988, 29.
(7) Sugiyama, H.; Ikeda, S.; Saito, I. J. Am. Chem. Soc. 1996, 118, 9994-
* To whom correspondence should be addressed. Tel.: (858) 784-7290.
Fax: (858) 784-7472. E-mail: floyd@scripps.edu.
(1) Kornberg, A.; Baker, T. A. DNA Replication, 2nd ed.; W. H. Freeman
and Co.: New York, 1992.
(2) Lutz, M. J.; Held, H. A.; Hottiger, M.; Hu¨bscher, U.; Benner, S. A.
Nucleic Acids Res. 1996, 24, 1308-1313.
(3) Horlacher, J.; Hottiger, M.; Podust, V. N.; Hu¨bscher, U.; Benner, S.
A. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 6329-6333.
(4) Piccirilli, J. A.; Krauch, T.; Moroney, S. E.; Benner, S. A. Nature
1990, 343, 33-37.
(5) Bain, J. D.; Switzer, C.; Chamberlin, A. R.; Benner, S. A. Nature
1992, 356, 537-539.
9995.
(8) Tor, Y.; Dervan, P. B. J. Am. Chem. Soc. 1993, 115, 4461-4467.
(9) Lutz, M. J.; Horlacher, J.; Benner, S. A. Bioorg. Med. Chem. Lett.
1998, 8, 499-504.
(10) Switzer, C.; Moroney, S. E.; Benner, S. A. J. Am. Chem. Soc. 1989,
111, 8322-8323.
(11) Switzer, C. Y.; Moroney, S. E.; Benner, S. A. Biochemistry 1993,
32, 10489-10496.
(12) Beaussire, J.-J.; Pochet, S. Nucleosides Nucleotides 1999, 18, 403-
410.
(13) Roberts, C.; Chaput, J. C.; Switzer, C. Chem., Biol. 1997, 4, 899-
908.
10.1021/ja0009931 CCC: $19.00 © 2000 American Chemical Society
Published on Web 07/28/2000

7622 J. Am. Chem. Soc., Vol. 122, No. 32, 2000
Wu et al.
polymerase that can replicate DNA containing these unnatural
H-bonding base pairs.2-4,9,11,16
In contrast, the design of the novel base pairs described herein
is based on hydrophobic packing and thus is not susceptible to
effects of tautomerization. By analogy to protein biochemistry,
the hydrophobic interactions between the bases are predicted
to be strong and selective for hydrophobic pair formation.17
Furthermore, mispairs between the unnatural and native bases
should be disfavored due to the energetic cost of desolvation
of the native base. In addition to stable and selective pairing,
the unnatural hydrophobic bases must be enzymatically repli-
cable. A DNA polymerase must be capable of recognizing the
hydrophobic substrates, both in template and as a triphosphate,
and efficiently incorporating them with high selectivity into
DNA. Finally, the polymerase must efficiently continue primer
extension after synthesis of the nascent unnatural base pair.
We have recently shown that the Watson-Crick H-bonds in
duplex DNA can be replaced with intrabase hydrophobic
interactions.18,19 A variety of hydrophobic nucleobases have been
shown to form base pairs in duplex DNA with a stability and
selectivity equivalent to or greater than those of native base
pairs.18-20 Moreover, several hydrophobic bases have been
identified which are efficiently incorporated into DNA with rates
approaching those of natural base pairs. Among these, the base
pair formed between the 7-azaindole (7AI) and isocarbostyril
(ICS) nucleosides possessed thermodynamic and kinetic proper-
ties that made it attractive for further investigation. The 7AI:
ICS base pair was only slightly less stable than an dA:dT base
pair (Tm ) 57.2 °C and 59.2 °C for 7AI:ICS and dA:dT,
respectively). The base pair was also thermally selective against
mispairing with natural bases; the most stable mispairs were
2.1 and 3.8 °C less stable for 7AI:ICS and dA:dT, respectively.
However, while the 7AI:ICS pair was 1.7 °C thermally selective
against the 7AI:7AI self-pair, which melted at 55.5 °C, it was
not selective against the ICS:ICS self-pair which melted at 59.3
°C. Therefore, it seemed that the interbase interactions between
7AI and ICS were not yet optimal for selective pairing in duplex
DNA.
Figure 1. Hydrophobic nucleosides.
Figure 2.
However, unlike the 7AI:ICS pair, neither of the self-pairs was
extended under any conditions examined. To optimize the
unnatural base pair, we have synthesized and characterized a
variety of derivatives of 7AI and ICS. The resulting base pairs
between the 7AI derivatives and the ICS derivatives have been
characterized thermodynamically in duplex DNA, and as
substrates for KF.
2. Results
In the context of enzymatic DNA replication, the 7AI:ICS
base pair also exhibited properties that made it attractive for
further optimization.19 The rates of exonuclease-deficient Kle-
now fragment (KF)-mediated insertion of dICSTP opposite 7AI
in the template strand, and d7AITP opposite ICS in the template
strand, were both reasonably efficient, with the value of kcat/
KM ) 2.7 × 105 and 1.8 × 105 M-1 min-1, respectively. While
these rates are roughly 100-fold slower than those typical of
native pair synthesis, the rates for mispairing with natural dNTPs
were significantly slower (less than 6 × 103 M-1 min-1),
resulting in reasonable fidelity. Preliminary results show that
the 7AI:ICS base pair is extendable with moderate efficiency
(104 M-1 min-1). However, as was the case with thermal
selectivity, the self-pairs constituted the major fidelity problem
for the 7AI:ICS base pair. The 7AI:7AI and ICS:ICS self-pairs
were synthesized nearly as efficiently as the 7AI:ICS pair.
2.1. Base Pair Design and Synthesis. In an effort to design
7AI and ICS analogues with improved thermodynamic and
kinetic properties, the 7AI and ICS derivatives shown in Figure
1 were synthesized. The methyl-substituted derivatives were
designed to probe the effects of minor groove (M7AI), major
groove (5MICS), and interbase (MICS) packing. Propynyl-
substituted derivatives (P7AI and PICS) were designed to
increase nucleobase hydrophobic surface area, and nitrogen-
substituted derivatives (ImPy and PP) were designed to modify
the electronic nature of the nucleobase without significant
structural perturbations.
The syntheses of nucleosides 1-6 involved two strategies
for stereoselective glycosidic bond formation. In all cases, the
bis-toluoyl-protected chloroglycoside 721 (Figure 2) served the
electrophile for condensation with each nucleobase.22 Depro-
tection under basic methanolic conditions22 afforded the respec-
tive nucleosides, which were converted to both nucleoside tri-
phosphates6 and 5′-trityl phosphoramidites for use in automated
DNA synthesis (Scheme 3).23 As previously reported, C1′-
stereochemical assignment of 1-6 was based on NOESY data.19
(14) Roberts, C.; Bandaru, R.; Switzer, C. J. Am. Chem. Soc. 1997, 119,
4640-4649.
(15) Robinson, H.; Gao, Y.-G.; Bauer, C.; Roberts, C.; Switzer, C.; Wang,
A. H.-J. Biochemistry 1998, 37, 10897-10905.
(16) Lutz, M. J.; Horlacher, J.; Benner, S. A. Bioorg. Med. Chem. Lett.
1998, 8, 1149-1152.
(17) Dill, K. A. Biochemistry 1990, 29, 7133-7155.
(18) McMinn, D. L.; Ogawa, A. K.; Wu, Y.; Liu, J.; Schultz, P. G.;
Romesberg, F. E. J. Am. Chem. Soc. 1999, 121, 11585-11586.
(19) Ogawa, A. K.; Wu, Y.; McMinn, D. L.; Liu, J.; Schultz, P. G.;
Romesberg, F. E. J. Am. Chem. Soc. 2000, 122, 3274-3287.
(20) Berger, M.; Ogawa, A. K.; McMinn, D. L.; Wu, Y.; Schultz, P. G.;
Romesberg, F. E. Angew. Chem., Int. Ed., in press.
(21) Takeshita, M.; Chang, C.-N.; Johnson, F.; Will, S.; Grollman, A.
P. J. Biol. Chem. 1987, 262, 10171-10179.
(22) Schweitzer, B. A.; Kool, E. T. J. Org. Chem. 1994, 59, 7238-
7242.
(23) Schweitzer, B. A.; Kool, E. T. J. Am. Chem. Soc. 1995, 117, 1863-
1872.

Optimization of Interbase Hydrophobic Interactions
J. Am. Chem. Soc., Vol. 122, No. 32, 2000 7623
Scheme 1a
Scheme 3a
a Conditions: (a) POCl3, 0 °C, then Bu3N-PPi; (b) DMTr-Cl; (c)
ClP(NiPr2)(OCH2CH2CN), NEt3.
of the hydrophobic bases, they were incorporated into the
complementary oligonucleotides, 5′-GCGATGXGTAGCG-3′
and 5′-CGCTACYCATCGC-3′, where X and Y are either
unnatural or natural bases. The relative stability of the base pairs,
as well as the mispairs with natural bases, was determined in
this sequence context by temperature-dependent absorption
changes as measured with a Cary 300 Bio UV/vis spectropho-
tometer. The absorption at 260 nm was monitored while the
samples were heated at a rate of 0.5 °C/min. We use the terms
“thermal stability” or “thermodynamic stability” of the X:Y base
pair to refer to the stability of the duplex as measured by the
duplex melting temperature (Tm) with oligonucleotides contain-
ing X and Y at the indicated positions. The stability difference
between a given base pair and the most stable mispair with
another base is referred to as thermal or thermodynamic
selectivity.
a Conditions: (a) NaH, then 7; (b) NaOMe; (c) ICl, CuI, Cl2Pd(Ph3)2,
NEt3, propyne, -78 °C to room temperature.
Scheme 2a
To facilitate the presentation of the data, the following results
are divided into two sections. The 7AI derivatives paired only
with ICS are discussed first, followed by an analysis of ICS
derivatives paired with 7AI. The base pairs, in which both 7AI
and ICS have been modified, are discussed only where
important. In each case, the thermal stability and selectivity of
the base pairs are first presented, followed by a discussion of
base pair recognition by KF.
2.2.1. Thermodynamic Properties of 7AI Derivatives. As
mentioned above, the 7AI:ICS unnatural base pair is 2.0 °C
less stable than a dA:dT base pair in the examined sequence
context.19 The thermal selectivity of the unnatural base pair (2.1
°C, relative to the most stable mispair) is also somewhat
compromised relative to that of dA:dT (3.8 °C).19 In an effort
to improve the interbase hydrophobic packing, the nucleoside
derivative, 6-methyl-7-azaindole (M7AI), was synthesized and
characterized. The methyl group is expected to occupy space
in the minor groove of the DNA duplex and may pack efficiently
opposite another hydrophobic base. When the unnatural base
is incorporated opposite ICS, PICS, MICS, or 5MICS, C6-
methyl substitution is found to increase duplex stability by 0.8-
2.7 °C (Table 1). Thus, the stability of the pairs formed between
M7AI and ICS derivatives is comparable with that of a native
dA:dT base pair in the same sequence context. However, the
mispairs between M7AI and the native bases are also stabilized
relative to the mispairs with 7AI by approximately the same
amount. This results in little change in thermal selectivity of
M7AI:ICS, as compared to that of 7AI:ICS. Furthermore, the
M7AI:M7AI self-pair is 2.5 °C more stable than the 7AI:7AI
a Conditions: (a) bis-TMS-acetamide, then 7, SnCl4; (b) NaOMe.
Purine analogues 1-4 followed from condensing the nucleo-
base sodium salts onto 7 and deprotection using the aforemen-
tioned conditions (Scheme 1).24 Specific to the synthesis of
nucleoside 4, 7-azaindole nucleoside 12 was first converted to
propynyl-substituted 13 prior to deprotection.18 Isocarbostyril
analogues 5 and 6 were synthesized via Vorbruggen glycosy-
lation methodology (Scheme 2) and deprotected as above.25
2.2. Thermodynamic and Kinetic Properties of 7AI:ICS
Base Pair Derivatives. So that we could evaluate the stability
(24) Seela, F.; Gumbiowski, R. Heterocycyles 1989, 29, 795-805.
(25) Niedballa, U.; Vorbruggen, H. J. Org. Chem. 1974, 39, 3654-3660.

7624 J. Am. Chem. Soc., Vol. 122, No. 32, 2000
Wu et al.
Table 1. Tm Values for Duplex-Containing 7AI Derivativesa
Table 2. Steady-State Kinetic Parameters for KF Exo-Mediated
Synthesis of DNA with 7AI, M7AI, and P7AI in the Templatea
5′-dGCGTACXCATGCG
3′-dCGCATGYGTACGC
5′-dTAATACGACTCACTATAGGGAGA
3′-dATTATGCTGAGTGATATCCCTCTXGTCA
X
Y
Tm (°C)
X
Y
Tm (°C)
nucleoside
template (X) triphosphate
kcat/KM
KM (µM) (M-1 min-1
)
A
7AI
T
59.2
55.5
57.2
57.6
59.7
62.5
57.3
52.5
50.5
51.5
48.5
58.0
58.7
58.6
60.5
59.7
60.0
54.2
52.2
52.5
49.5
56.3
56.8
58.6
59.3
61.2
57.9
51.2
50.3
50.2
45.7
G
ImPy
C
61.8
52.7
54.7
57.3
59.7
60.0
56.6
52.3
50.0
51.0
46.3
51.3
56.3
56.3
58.5
57.2
56.3
50.0
50.8
50.5
46.3
k
cat (min-1
)
7AI
ICS
PICS
MICS
PIM
5MICS
A
T
G
C
M7AI
ICS
PICS
MICS
PIM
5MICS
A
T
G
C
P7AI
ICS
PICS
MICS
PIM
5MICS
A
T
G
C
ImPy
ICS
PICS
MICS
PIM
5MICS
A
T
G
C
PP
ICS
PICS
MICS
PIM
5MICS
A
T
G
C
7AI
7AI
M7AI
ICS
PICS
MICS
5MICS
A
G
C
T
M7AI
7AI
ICS
PICS
MICS
5MICS
A
G
C
T
P7AI
ICS
PICS
MICS
5MICS
A
G
C
T
7.6 ( 0.4
1.5 ( 0.1
7.4 ( 0.3
2.9 ( 0.2
22 ( 1
34 ( 5
124 ( 23
27 ( 3
2.2 × 105
1.2 × 104
2.7 × 105
3.9 × 105
1.5 × 106
1.3 × 105
5.9 × 103
1.6 × 103
5.5 × 103
1.6 × 103
6.3 × 103
7.6 × 104
1.2 × 105
1.8 × 105
1.1 × 106
6.3 × 104
3.9 × 103
1.5 × 103
1.0 × 103
2.2 × 103
1.4 × 106
1.1 × 105
3.4 × 105
4.4 × 105
2.9 × 104
1.1 × 104
4.8 × 104
4.6 × 106
2.0 × 104
7.5 ( 2.0
15 ( 1
3.3 ( 0.2
26 ( 2.0
54 ( 10
49 ( 13
31 ( 11
0.32( 0.02
0.08( 0.01
0.17 ( 0.02
0.23 ( 0.05 146 ( 40
M7AI
PP
M7AI
0.42 ( 0.03
2.2 ( 0.2
3.4 ( 0.1
0.9 ( 0.1
20 ( 1
67 ( 12
29 ( 8
29 ( 2
5.1 ( 1.2
19 ( 2
2.0 ( 0.1
0.11 ( 0.01
0.06 ( 0.01
0.03 ( 0.01
0.17 ( 0.02
11.2 ( 0.1
2.9 ( 0.1
1.8 ( 0.1
8.0 ( 0.3
0.9 ( 0.2
0.41 ( 0.02
1.1 ( 0.1
2.3 ( 0.1
1.1 ( 0.1
32 ( 2
28 ( 11
38 ( 10
30 ( 10
76 ( 24
8.1 ( 0.3
26 ( 3
P7AI
P7AI
5.3 ( 0.9
18 ( 2
31 ( 15
36 ( 8
23 ( 4
0.5 ( 0.3
56 ( 20
a See text for experimental details.
a See text for experimental details.
significantly destabilized, by 4.2 °C relative to 7AI:7AI,
providing good thermal orthogonality for PP against all possible
mispairs.
self-pair, which further limits the thermal orthogonality of the
M7AI nucleobase.
The effect of C3-propynyl substitution of the azaindole ring
was examined with propynyl 7-azaindole (P7AI). The duplex
containing the P7AI:ICS base pair is destabilized 0.4 °C relative
to the 7AI:ICS base pair. All of the mispairs with native bases
are also destabilized (by 0.2-2.8 °C). In the case of 7AI, the
most stable mispairs are 7AI:dA and 7AI:dG. With propynyl
substitution, both of these mispairs are more destabilized than
the pairs with ICS or ICS derivatives. Therefore, compared to
7AI, P7AI is more thermally orthogonal relative to mispairing
with native bases. However, the P7AI nucleobase is less
orthogonal relative to self-pairing, as the P7AI:P7AI self-pair
is stabilized by 0.8 °C relative to the 7AI self-pair.
2.2.2. Kinetic Properties of 7AI Derivatives. The 7AI
derivative nucleobases were also evaluated as substrates for KF.
Initial velocities were determined by literature methods using
[γ-32P]primer extension reactions with varying concentrations
of nucleoside triphosphates.26 The reactions were analyzed by
polyacrylamide gel electrophoresis and subsequent Phospho-
rimager (Molecular Dynamics) analysis to quantify gel band
intensities corresponding to the extended primer. The measured
velocities were plotted against the concentration of dNTP and
fit to the Michaelis-Menten equation. Unnatural nucleobases
were assayed both in template DNA and as incoming nucleoside
triphosphates. Steady-state kinetic parameters for single nucle-
otide incorporation (apparent kcat and apparent KM) are reported
in Tables 2 and 3.
As mentioned above, the7AI:ICS base pair was synthesized
by KF with reasonable efficiency, while mispairs with native
bases were synthesized significantly slower.19 However, the
synthesis of 7AI:ICS was significantly less efficient than that
for native base pairs. Therefore, it is desirable to further optimize
the rate at which KF synthesizes the unnatural base pair.
Increased fidelity both against mispairing with native bases, and
against self-pairing, is also necessary to make DNA replication
practical.
Isostructural modifications of the electronic nature of the
nucleobases were examined by substitution of CH groups with
N atoms (aza-substitution). Specifically, the imidizopyridine
(ImPy) and pyrrolopyrizine (PP) nucleosides were examined.
ImPy:ICS and PP:ICS are both less stable than 7AI:ICS, by
2.5 and 0.9 °C, respectively (Table 1). The mispairs are also
destabilized with ImPy, by 0.2-0.5 °C for dA, dT, and dG,
and by 2.2 °C for dC. This decrease in mispair stability is
perhaps surprising, at least in the case of ImPy:dC and ImPy:
dT, considering the more purine-like ring structure of ImPy
relative to that of 7AI. For PP, the mispair with dT is slightly
stabilized, while those with dC, dA, and dG are destabilized by
1.0-2.5 °C, again relative to the same mispairs with 7AI. The
low stability of these mispairs, and the reasonable stability of
the PP:ICS and PP:MICS base pairs, give this azaindole
analogue increased thermodynamic orthogonality relative to
mispairing with native bases. Moreover, the PP:PP self-pair is
The effect of C6-methyl substitution on enzymatic synthesis
was examined first. With M7AI in template, the efficiency of
dM7AITP incorporation is low (kcat/KM ) 6.3 × 103 M-1 min-1
,
(26) Goodman, M. F.; Creighton, S.; Bloom, L. B.; Petruska, J. Crit.
ReV. Biochem. Mol. Biol. 1993, 28, 83-126.

Optimization of Interbase Hydrophobic Interactions
J. Am. Chem. Soc., Vol. 122, No. 32, 2000 7625
Table 3. Steady-State Kinetic Parameters for KF Exo-Mediated
Table 5. Steady-State Kinetic Parameters for KF Exo-Mediated
Synthesis of DNA with ImPy and PP in the Templatea
Synthesis of DNA with ICS and PICS in the Templatea
5′-dTAATACGACTCACTATAGGGAGA
3′-dATTATGCTGAGTGATATCCCTCTXGTCA
5′-dTAATACGACTCACTATAGGGAGA
3′-dATTATGCTGAGTGATATCCCTCTXGTCA
nucleoside
template (X) triphosphate
kcat/KM
nucleoside
template (X) triphosphate
kcat/KM
k )
cat (min-1
KM (µM) (M-1 min-1
)
k
cat (min-1
)
KM (µM) (M-1 min-1
)
ICS
ICS
PICS
7AI
M7AI
P7AI
ImPy
PP
A
G
C
T
ICS
PICS
7AI
M7AI
P7AI
ImPy
PP
A
G
C
T
3.6 ( 0.1
2.6 ( 0.4
10.6 ( 0.5
1.6 ( 0.2
28.8 ( 1.6
1.3 ( 0.1
4.9 ( 0.2
0.15 ( 0.01
0.28 ( 0.02
0.03 ( 0.01
1.2 ( 0.1
1.2 ( 0.1
0.90 ( 0.04 3.7 ( 0.7
4.5 ( 0.1
0.88 ( 0.04
43 ( 1
57 ( 6
33 ( 11
60 ( 15
204 ( 60
15 ( 2
6.3 × 104
7.8 × 104
1.8 × 105
7.8 × 103
1.9 × 106
4.1 × 104
4.1 × 105
2.1 × 103
3.4 × 103
8.8 × 102
5.1 × 103
4.8 × 104
2.4 × 105
2.1 × 105
1.6 × 104
1.0 × 107
5.2 × 104
4.7 × 105
1.8 × 103
1.7 × 103
1.8 × 103
1.2 × 10xt
ImPy
ImPy
ICS
PICS
MICS
5MICS
A
G
C
T
PP
ICS
PICS
MICS
5MICS
A
G
C
T
2.0 ( 0.1
3.3 ( 0.5
2.4 ( 0.3
30 ( 1
5.5 ( 0.2
1.6 ( 0.1
109 ( 11
19 ( 6
2.7 ( 1.4
14 ( 1
28 ( 3
50 ( 7
1.8 × 104
1.7 × 105
8.9 × 105
2.1 × 106
2.0 × 105
3.2 × 104
9.0 × 102
1.4 × 104
5.7 × 103
1.7 × 105
1.4 × 105
5.7 × 105
2.0 × 106
8.9 × 104
2.2 × 104
1.3 × 104
9.3 × 104
8.7 × 103
32 ( 15
12 ( 2
0.12 ( 0.01 134 ( 27
73 ( 10
83 ( 12
34 ( 19
234 ( 83
25 ( 3
2.6 ( 0.2
190 ( 50
0.89 ( 0.05 157 ( 19
PP
2.5 ( 0.1
3.5 ( 0.1
2.0 ( 0.1
20 ( 1
2.5 ( 0.1
1.1 ( 0.1
0.54 ( 0.04
2.5 ( 0.2
0.85 ( 0.04
15 ( 5
25 ( 3
3.5 ( 0.5
10 ( 1
28 ( 3
49 ( 7
41 ( 6
27 ( 12
98 ( 11
PICS
21 ( 2
56 ( 9
4.1 ( 0.1
25 ( 5
6 ( 2
92 ( 30
69 ( 22
1.3 ( 0.1
2.8 ( 0.2
0.17 ( 0.07
0.12 ( 0.04
0.22 ( 0.12 123 ( 81
0.89 ( 0.15
a See text for experimental details.
76 ( 36
Table 4. Tm Values for Duplex-Containing ICS Derivativesa
a See text for experimental details.
5′-dGCGTACXCATGCG
3′-dCGCATGYGTACGC
Table 6. Steady-State Kinetic Parameters for KF Exo-Mediated
Synthesis of DNA with MICS and 5MICS in the Templatea
X
Y
Tm (°C)
X
Y
Tm (°C)
A
G
ICS
T
C
ICS
7AI
M7AI
A
T
G
C
MICS
A
59.2
61.8
59.3
56.5
58.5
55.1
53.0
51.0
52.2
62.3
53.6
55.1
54.8
54.8
PICS
PICS
7AI
M7AI
P7AI
PP
A
T
62.6
56.8
59.7
58.8
56.0
55.5
53.7
54.5
51.4
61.2
55.7
55.5
55.5
54.3
5′-dTAATACGACTCACTATAGGGAGA
3′-dATTATGCTGAGTGATATCCCTCTXGTCA
nucleoside
template (X) triphosphate
kcat/KM
k
cat (min-1
)
KM (µM) (M-1 min-1
)
MICS
MICS
ICS
7AI
M7AI
P7AI
ImPy
PP
A
G
C
T
5MICS
ICS
7AI
M7AI
P7AI
ImPy
PP
A
G
C
T
4.6 ( 0.1
2.2 ( 0.2
7.0 ( 0.3
1.0 ( 0.1
19 ( 1
14 ( 1
37 ( 14
44 ( 5
57 ( 15
6.2 ( 1.2
18 ( 4
5.9 ( 1.6
15 ( 2
48 ( 6
43 ( 15
80 ( 7
ndb
3.3 × 105
5.9 × 104
1.8 × 105
1.8 × 104
3.1 × 106
8.3 × 104
1.4 × 106
2.4 × 104
5.8 × 103
6.3 × 103
2.0 × 104
e1.0 × 103
1.4 × 104
6.9 × 104
7.3 × 103
1.4 × 106
2.7 × 104
3.2 × 105
e1.0 × 103
e1.0 × 103
e1.0 × 103
e1.0 × 103
G
C
MICS
5MICS
5MICS
A
T
G
C
1.5 ( 0.1
8.4 ( 0.7
T
G
C
0.36 ( 0.02
0.28 ( 0.02
0.27 ( 0.03
1.6 ( 0.1
ndb
1.6 ( 0.2
6.9 ( 0.1
0.9 ( 0.1
23 ( 2
a See text for experimental details.
5MICS
Table 2). Therefore, despite the high thermal stability of the
M7AI:M7AI self-pair, it is not efficiently synthesized by KF,
making M7AI a promising nucleobase to pair with ICS
derivatives. The incorporation efficiency of dICSTP opposite
M7AI is within a factor of 2.5 of that with 7AI in the template
(kcat/KM ) 2.3 × 105 and 7.6 × 104 M-1 min-1 for 7AI and
M7AI in template, respectively). Native triphosphates are
incorporated opposite M7AI in the template with rates of
approximately 2 × 103 M-1 min-1. Therefore, KF selectively
incorporates dICSTP opposite M7AI in the template by a factor
of 20-80-fold relative to incorporation of native triphosphates.
This selectivity is 2-fold reduced relative to that observed with
7AI in the template (dICSTP is inserted opposite 7AI 40-140-
fold faster than any native triphosphate).
112 ( 42
100 ( 5
124 ( 27
16 ( 4
26 ( 10
7.3 ( 1.0
ndb
0.7 ( 0.1
2.3 ( 0.1
ndb
ndb
ndb
ndb
ndb
ndb
ndb
a See text for experimental details. b Rates too slow for determination
of kcat and KM independently.
1.6 × 105 to 2.8 × 103 M-1 min-1). The decreased rates for
insertion result from both kcat and KM effects.
Differences between M7AI and 7AI are more pronounced
when the unnatural base is present as a triphosphate. The
triphosphate dM7AITP is incorporated 9-23-fold less efficiently
than d7AITP opposite ICS and its derivatives in the template
(Tables 5 and 6). However, dM7AITP is inserted even less
efficiently opposite native bases relative to d7AITP (Table 7).
For example, the incorporation efficiency of dM7AITP opposite
dA is reduced 50-fold relative to d7AITP opposite dA (from
With P7AI in the template, the incorporation efficiency of
dICSTP is reduced by approximately 2-fold relative to that with
7AI in the template (Table 2). Conversely, native triphosphates
are incorporated more efficiently opposite P7AI than opposite
7AI, by 2-30-fold for dATP, dTTP, and dGTP. However, the
incorporation of dCTP opposite P7AI is approximately 1000-
fold more efficient than that opposite 7AI in the template. This
remarkable, and selective, enhancement in the efficiency of

7626 J. Am. Chem. Soc., Vol. 122, No. 32, 2000
Wu et al.
Table 7. Steady-State Kinetic Parameters for KF Exo-Mediated
dATP, dCTP, and dTTP, and 2-fold slower for dGTP). A 15-
fold increase in kcat for dCTP insertion is offset by a 6-fold
increase in KM(dCTP). The insertion of dATP is only 6-fold
slower than the insertion of dICSTP, limiting the orthogonality
of ImPy. Insertion of dImPyTP opposite ICS proceeds with an
efficiency of 4.1 × 104 M-1 min-1, which is 4-fold reduced
relative to the insertion of d7AITP (1.8 × 105 M-1 min-1). The
insertion efficiency of dImPyTP opposite dC is equal to that of
d7AITP opposite dC, while insertion of dImPyTP opposite dG,
dA, and dT is 2-7-fold slower by comparison. The ImPy:ImPy
self-pair is synthesized by KF 12-fold less efficiently than the
7AI:7AI self-pair (Table 7).
Synthesis of DNA with A, G, C, and T in the Templatea
5′-dTAATACGACTCACTATAGGGAGA
3′-dATTATGCTGAGTGATATCCCTCTXGTCA
nucleoside
template (X) triphosphate
kcat/KM
k
cat (min-1
)
KM (µM) (M-1 thrmin-1
)
A
T
7AI
M7AI
P7AI
ImPy
PP
163 ( 7
3.5 ( 1.0
48 ( 10
4.7 × 107
1.6 × 105
2.8 × 103
2.4 × 106
2.3 × 104
5.1 × 105
6.0 × 104
2.0 × 104
2.6 × 105
1.3 × 104
2.1 × 104
e1.0 × 103
2.9 × 105
4.5 × 103
7.6 × 104
1.8 × 104
9.7 × 103
7.8 × 104
3.1 × 103
1.9 × 104
e1.0 × 103
7.2 × 105
1.8 × 104
5.1 × 105
2.4 × 104
4.0 × 105
1.3 × 105
3.9 × 103
4.1 × 104
2.8 × 103
8.9 × 105
1.6 × 104
2.6 × 105
4.8 × 104
3.0 × 105
2.8 × 105
1.7 × 104
7.5 ( 0.8
0.28 ( 0.02 100 ( 20
21 ( 1
8.8 ( 1.7
70 ( 10
11 ( 2
1.6 ( 0.1
5.6 ( 0.2
3.0 ( 0.3
ICS
50 ( 16
PICS
MICS
5MICS
7AI
M7AI
P7AI
ImPy
PP
0.40 ( 0.10 20 ( 10
The rate of KF-mediated insertion of dICSTP opposite PP in
the template is reasonably efficient (kcat/KM ) 1.4 × 105 M-1
min-1), but approximately 2-fold slower than the rate of insertion
of dICSTP opposite 7AI. The insertions of triphosphates dGTP,
dATP, and dTTP are slightly more efficient opposite PP than
opposite 7AI (by 4-8-fold). The insertion of dCTP opposite
PP is 17-fold more efficient than the insertion of dCTP opposite
7AI (Table 3).
9.3 ( 0.2
36 ( 2
0.89 ( 0.02 69 ( 5
0.63 ( 0.02 30 ( 3
G
C
T
ndb
ndb
26 ( 2
7.6 ( 0.2
0.27 ( 0.01 60 ( 9
0.71 ( 0.06 9.3 ( 2.6
0.48 ( 0.02 27 ( 3
0.09 ( 0.01 9.3 ( 3.6
2.25 ( 0.03 29 ( 1
0.16 ( 0.01 51 ( 11
ICS
PICS
MICS
5MICS
7AI
M7AI
P7AI
ImPy
PP
The triphosphate dPPTP is inserted opposite ICS in the
template approximately 2-fold faster than d7AITP is inserted
opposite ICS. Similar to the behavior of PP in the template,
insertion of dPPTP opposite native templates dA, dG, and dT
is slightly faster than the insertion of d7AITP (3-6-fold), while
the insertion of dPPTP opposite dC is 27-fold faster than that
of d7AITP (Table 7). Therefore, inclusion of the nitrogen at
position 4 of the azaindole ring selectively favors the synthesis
of the mispair between the unnatural base and natural bases
(especially cytosine) in both template-triphosphate combina-
tions. Despite the increased rates for the incorporation of dPPTP
opposite dC, the insertion of the PP triphosphate is still 2 orders
of magnitude slower than the insertion of dGTP. The self-pair
PP:PP is synthesized slightly slower than the 7AI:7AI self-pair,
1.7 ( 0.1
88 ( 4
ndb
ndb
15.9 ( 0.2
22 ( 1
0.73 ( 0.04 40 ( 6
5.0 ( 0.2
1.7 ( 0.2
9.9 ( 1.0
72 ( 10
ICS
PICS
MICS
5MICS
7AI
M7AI
P7AI
ImPy
PP
1.05 ( 0.04 2.6 ( 0.7
4.7 ( 0.2
35 ( 3
0.54 ( 0.04 140 ( 23
4.6 ( 0.1 113 ( 7
0.28 ( 0.02 100 ( 20
21.4 ( 0.7
1.2 ( 0.1
4.9 ( 0.2
24 ( 2
75 ( 12
19 ( 2
ICS
4.9 ( 0.2 103 ( 13
2.80 ( 0.10 9.4 ( 2.0
PICS
MICS
5MICS
with an efficiency of 1.7 × 105 M-1 min-1
.
15 ( 1
54 ( 5
2.1 ( 0.10 126 ( 15
2.2.3. Thermodynamic Properties of ICS Derivatives. To
further optimize the unnatural base pair we examined the
stability of base pairs formed with the ICS derivatives shown
in Figure 1. As reported previously, C7-propynyl substitution
marginally stabilizes the unnatural pair 7AI:PICS relative to 7AI:
ICS (Tm ) 57.6 and 57.2 °C, respectively, Table 4).19 The
mispairs between PICS and dA or dT are both slightly more
stable than the mispairs between ICS and dA or dT (by 0.4-
0.7 °C). The PICS mispair with dG is 1.8 °C more stable, while
the mispair with dC is 0.8 °C less stable, relative to the same
mispairs with ICS. Relative to ICS, PICS stabilizes the unnatural
pairs with each 7AI derivative slightly more than it stabilizes
the most stable of the mispairs (PICS:dA). Therefore, PICS is
slightly more thermodynamically orthogonal to the native bases
than is ICS. The remarkable stability of the PICS:PICS self-
pair (Tm ) 62.6 °C) limits the thermal orthogonality of the 7AI:
PICS unnatural base pair.
a See text for experimental details. b Rates too slow for determination
of kcat and KM independently.
dCTP incorporation upon propynyl substitution results from a
14-fold increase in apparent kcat and a 62-fold decrease in
apparent KM(dCTP).
The triphosphate, dP7AITP is inserted opposite ICS with high
efficiency (1.9 × 106 M-1 min-1, Table 5). Relative to the
incorporation of d7AITP opposite ICS, dP7AITP is inserted 10-
fold more efficiently due to both an increase in kcat (3-fold)
and a decrease in KM (4-fold). However, the triphosphate
dP7AITP is also inserted with increased efficiency opposite all
of the native templates, relative to d7AITP. Insertion opposite
purines (dA and dG) is approximately 15-fold more efficient,
while insertion opposite pyrimidines (dT and dC) is 20-40-
fold more efficient. The self-pair P7AI:P7AI is also synthesized
with increased efficiency (by 6-fold), relative to 7AI:7AI, again
limiting the usefulness of this nucleobase for expansion of the
genetic alphabet.
Isostructural alterations of the electronic nature of the
nucleobase were also examined with respect to enzymatic DNA
synthesis. The effect of N3 substitution was determined by
comparing the rate for dICSTP incorporation opposite 7AI and
ImPy. Insertion of dICSTP opposite ImPy in the template
proceeds with the same efficiency (1.7 × 107 M-1 min-1) as
that of dICSTP opposite 7AI. Native triphosphates are inserted
opposite ImPy with efficiencies that are all less than 3.2 × 104
M-1 min-1 (2-5-fold faster than insertion opposite 7AI for
The effect of C3-methyl substitution was examined with the
isocarbostyril derivative MICS. The added methyl group of
MICS has a pronounced effect on base pair stability, with 7AI:
MICS stabilized by 2.5 °C relative to the 7AI:ICS base pair
(Tm ) 59.7 °C relative to 57.2 °C, Table 4). The mispairs formed
between MICS and dT, dG, and dC are also stabilized by 2.1-
3.8 °C, while the mispair with dA is destabilized by 1.5 °C.
The stabilities of the most stable mispair formed between ICS
or MICS and a native base are identical (Tm ) 55.1 °C for ICS:
dA and MICS:dT). However, 7AI:MICS is more stable than
7AI:ICS, and thus MICS is more thermodynamically orthogonal
than ICS relative to the native bases. In fact, the 7AI:MICS

Optimization of Interbase Hydrophobic Interactions
J. Am. Chem. Soc., Vol. 122, No. 32, 2000 7627
base pair is both more stable than a dA:dT base pair (Tm
)
proximately 5-fold more efficiently than dICSTP. This results
primarily from a 3-5-fold increase in kcat, along with an up to
2-fold decrease in KM for dMICSTP relative to that for dICSTP.
Insertions of dMICSTP opposite dA, dC, and dT were catalyzed
with large values of kcat/KM, approaching 3 × 105 M-1 min-1
(Table 7). However, this rate is 2 orders of magnitude lower
than the rate at which KF pairs the same templates with their
native partners. Therefore, incorporation of the unnatural base
at these positions does not compete with native synthesis.
Enzymatic synthesis of base pairs containing 5MICS was also
examined. The 5MICS:5MICS self-pair is synthesized with low
efficiency (less than 1 × 103 M-1 min-1, Table 5). However,
the generally slow rates of unnatural base pair synthesis with
5MICS in the template or as the triphosphate (6.9 × 104 and
1.3 × 105 M-1 min-1, respectively with 7AI) limit the practical
utility of 5MICS.
59.2 °C for dA:dT) and more selective against mispairing with
native bases (thermal selectivity, relative to the most stable
mispair, is 4.6 and 3.8 °C for 7AI:MICS and dA:dT, respec-
tively). 7AI:MICS is more stable than the 7AI:7AI self-pair (by
4.2 °C) but less stable than the MICS:MICS self-pair (by 2.6
°C).
In addition to the effects of methyl substitution at C3, the
effects of methyl substitution at C5 of the isocarbostyril core
(5MICS) were examined. Methyl substitution at the C5 position
is significantly less stabilizing than that at the C3 position (Table
4). The stability of the 7AI:5MICS base pair is equal to that of
the 7AI:ICS base pair. Surprisingly, however, the mispairs
between 5MICS and the native bases are all stabilized by the
inclusion of the C5-methyl group. The mispair with dA is
stabilized by 0.6 °C, relative to the ICS mispairs, while the
mispairs with dC, dT, and dG are stabilized by 2.1-4.5 °C.
The C5-methyl substitution also stabilizes the self-pair by 1.9
°C. The increased stabilities of the mispairs and the self-pair
make 5MICS less thermally orthogonal than ICS.
3. Discussion
The practical replication of DNA containing three base pairs
requires that the rate for unnatural pair synthesis approach those
rates characteristic of native pair synthesis. Fidelity of replication
must also be high, especially with respect to self-pairing. Any
improvements in enzymatic replication must be accomplished
without significantly compromising base pair stability or thermal
selectivity in duplex DNA.
Hydrophobic bases, such as derivatives of 7AI:ICS that are
efficiently recognized by KF, and recognized with good fidelity,
are potentially realistic candidates for expansion of the genetic
alphabet. Modifications of the 7AI:ICS base pair were found
which improved both the efficiency and the fidelity of synthesis.
Examination of the data also reveals much about the general
stability of hydrophobic bases in duplex DNA, as well as the
recognition of these bases by KF during DNA synthesis.
3.1. Alkyl and Aza Substituent Effects. 3.1.1. Effect of
Methyl Substitution. The optimization of interstrand hydro-
phobic packing was examined by generating methyl-substituted
derivatives of 7AI and ICS. The 7-azaindole analogue M7AI
has a C6-methyl group that is expected to reside in the minor
groove. One ICS analogue, MICS, has a C3-methyl group that
is expected to be positioned in the interface with the pairing
base. Another ICS analogue, 5MICS, has a C5-methyl group
that is expected to be positioned in the major groove.
2.2.4. Kinetic Properties of ICS Derivatives. As reported
previously, PICS has been examined as a substrate for KF.18,19
With PICS in the template, there was little difference in
efficiency of d7AITP incorporation relative to that with ICS in
the template (Table 5). The rates for insertion of native
triphosphates opposite PICS are also relatively unchanged, as
compared to those with ICS in the template. There is a slight
increase in insertion efficiency for dCTP and dTTP, and a slight
decrease in efficiency for insertion of dGTP and dATP opposite
PICS in the template. When present as the triphosphate, the
dPICSTP propynyl group again had little effect on incorporation
efficiencies opposite 7AI (Table 2). The propynyl group had
larger effects on the efficiency of incorporation of the unnatural
triphosphate opposite native templates (Table 7). In each case,
the added propynyl group resulted in a decrease in kcat (1.6-
8-fold) and a relatively larger decrease in KM (2-30-fold).
Opposite dA and dG, dPICSTP was incorporated slower than
dICSTP (by 2- and 3-fold, respectively), and opposite dT and
dC, dPICSTP was incorporated 6- and 17-fold faster than
dICSTP. The PICS:PICS self-pair is synthesized almost 4-fold
more efficiently than the ICS:ICS self-pair, limiting the kinetic
orthogonality of PICS.
C6-Methyl substitution of 7AI (M7AI) and C3-methyl
substitution of ICS (MICS) strongly stabilized both pairing with
other unnatural bases and mispairing with native bases. A single
exception was the mispair between MICS:dA, which was
destabilized by 1.5 °C relative to ICS:dA. Methyl substitution
at the C5 position of ICS (5MICS) weakly stabilized pairing
with other hydrophobic bases but stabilized mispairing with
native bases more strongly. All of the methyl-substituted
derivatives were found to form more stable self-pairs than 7AI
or ICS.
The data imply that inclusion of a methyl group in the minor
groove, major groove, or interbase interface methyl groups leads
to specific interbase interactions in addition to increased buried
hydrophobic surface area. This follows from the observation
that although methyl substitution in general increased the
stability of all base pairs, the extent of stabilization was not
uniform. For example, the greatest increase in stabilization
afforded by the addition of the C3-methyl group of MICS was
observed when the unnatural base was incorporated opposite
less hydrophobic dG and dC.27 In contrast, there was a
The effects of the C3-methyl group on KF-mediated DNA
synthesis were examined with MICS in the template and d7AITP
(Table 6). Small differences are found upon modification of
unnatural base in the template. However, opposite MICS, the
insertion of both dImPyTP (8.3 × 104 M-1 min-1) and dPPTP
(1.4 × 106 M-1 min-1) is more efficient than that opposite other
ICS analogues in the template. For example, relative to d7AITP,
dPPTP is inserted 8-fold more efficiently opposite MICS. The
triphosphate d7AITP is inserted with the same efficiency
opposite both ICS and MICS in the template (1.8 × 105 M-1
min-1). The rates for insertion of the native triphosphates
opposite MICS are 2-11-fold more efficient than those for
insertion opposite ICS. The largest increase observed is in the
incorporation of dATP, which proceeds with an efficiency of
2.4 × 104 M-1 min-1
.
For a triphosphate, incorporation of dMICSTP by KF opposite
the 7AI analogue bases in the template is 4-14-fold more
efficient than that of dICSTP opposite the same templates
(Tables 2 and 3). The greatest increase is observed for the
incorporation of dMICSTP opposite PP, which proceeds with
an efficiency of 2 × 106 M-1 min-1. With templates containing
native bases, dMICSTP is also incorporated by KF ap-
(27) Shih, P.; Pedersen, L. G.; Gibbs, P. R.; Wolfenden, R. J. Mol. Biol.
1998, 280, 421-430.

7628 J. Am. Chem. Soc., Vol. 122, No. 32, 2000
Wu et al.
significant destabilization opposite dA, the most hydrophobic
of the native bases.27 Selective destabilization of the mispair
with dA is particularly advantageous for the orthogonality of
MICS and shows a clear improvement over the highly stable
ICS:dA mispair.
propynyl substitution (where only one base of the pair has a
propynyl group, i.e., ICS:PICS) was destabilizing.19 However,
symmetrical propynyl substitution (i.e., PICS:PICS) was strongly
stabilizing.19 This trend was also apparent in the data discussed
above. The base pair between 7AI and ICS was stabilized by
-0.4 to 0.4 °C upon addition of a single propynyl group to
either unnatural base (i.e., 7AI:PICS or P7AI:ICS). However,
with symmetrical propynyl substitution (P7AI:PICS, P7AI:P7AI,
or PICS:PICS), the base pairs were stabilized by 0.8-3.3 °C.
Similar to the effects of methyl group modification, the
addition of a propynyl group to the unnatural base in the
template resulted in only modest effects on base pair recognition
by KF. Exceptions were the efficient incorporation of dTTP,
dGTP, and especially dCTP opposite P7AI, relative to 7AI in
the template. In these cases, the added propynyl group increased
binding of the native triphosphates (by 2-60-fold) and catalytic
turnover (by 5-14-fold), resulting in a large increase in the
incorporation efficiency of these dNTPs opposite P7AI, as
compared to 7AI (13-800-fold). In fact, the rate of dCTP
incorporation opposite P7AI (4.6 × 106 M-1 min-1) was only
10-fold slower than the rate of dG:dC synthesis in the same
sequence context. In the context of KF-mediated DNA synthesis,
P7AI proved not to be orthogonal to native templates, but rather
to be an efficient dG mimic. As this effect is not reflected in
the thermodynamic data (in fact, dC:P7AI is 2.8 °C less stable
than dC:7AI), it seems that the efficient insertion of dCTP
opposite P7AI is mediated by interactions specific to the
polymerase or the insertion transition state.
The addition of a propynyl group to the isocarbostyril
triphosphate (dPICSTP) also had only modest effects, with
efficiencies that were within a factor of 5 relative to dICSTP,
resulting primarily from tighter binding of the triphosphate with
a propynyl group. However, addition of a propynyl group to
7AI had larger effects. Opposite both unnatural and natural
templates, dP7AITP is inserted 10-50-fold more efficiently than
is d7AITP. This increased efficiency resulted from a combina-
tion of kcat and KM effects. The largest increase was for the
incorporation of dP7AITP opposite PICS, the only unnatural
template to also have a major groove propynyl group. It is
tempting to speculate that increased kinetic efficiency might
be related to the thermal stability characteristic of base pairs
that are symmetrically substituted with propynyl groups, as
discussed above. This would imply that the interactions which
stabilize the symmetrically substituted base in duplex DNA are
significantly developed in the transition state for the incorpora-
tion of an unnatural triphosphate bearing a propynyl group.
Unlike P7AI in the template, dP7AITP is not a specific mimic
of dGTP, being incorporated less efficiently opposite dC than
opposite either dA or dT.
The discovery that H-bonds are not an absolute requirement
for enzymatic DNA synthesis28-30 has strengthened the argu-
ment that the fidelity of DNA synthesis derives, at least in part,
from a polymerase-mediated steric matching of the pairing
bases.31-33 Therefore, optimization of interstrand hydrophobic
packing, by generating methyl-substituted derivatives of 7AI
and ICS, might allow for the selective optimization of correct
unnatural pair synthesis relative to the synthesis of mispairs.
The major fidelity problem of the parental 7AI:ICS base pair
resulted from the 7AI:7AI and ICS:ICS self-pairs.19 The addition
of either a major groove (5MICS) or a minor groove (M7AI)
methyl group decreased the rate of self-pair synthesis. The
M7AI:M7AI and 5MICS:5MICS self-pairs were synthesized 37-
to >50-fold less efficiently than the 7AI:M7AI and ICS:ICS
self-pairs, respectively. Despite the slow rates of self-pair
synthesis, M7AI and 5MICS are limited in their practical utility
because KF does not efficiently incorporate d5MICSTP opposite
any of the hydrophobic bases examined (see below). In contrast,
the C3-methyl substitution of ICS increased the rate of self-
pair formation (MICS:MICS) by a factor of 5, relative to that
of the ICS:ICS self-pair. However, the relatively efficient
formation of the MICS:MICS self-pair does not preclude the
use of MICS for heteropair formation with 7AI derivatives,
including PP (see below), due to the high efficiency with which
the heteropairs are formed.
The shape of the unnatural base also affected the synthesis
of heteropairs. However, the nature of the base in the template,
natural or unnatural, had only small effects on the rates of
insertion of a given triphosphate. Addition of a methyl group
typically changed the rate for the insertion of a natural or an
unnatural triphosphate by less than 5-fold. The rates of unnatural
pair synthesis were more sensitive to modification of the
triphosphate. The largest effects were observed when the
isocarbostyril ring was modified at the C3 position (dMICSTP).
Relative to dICSTP, the rates for dMICSTP insertion opposite
7AI analogue templates increased by 6-15-fold, resulting in
very efficient rates of 106 M-1 min-1. The rates for incorporation
of dMICSTP opposite native templates were also increased (by
4-6-fold), but not to the extent that they were competitive with
insertion of a correct, native triphosphate. The C5-methyl group
of d5MICSTP resulted in a small decrease in the rate of
enzymatic pairing with 7AI derivatives (by 2-4-fold) or native
templates (by 3-6-fold). A significant decrease in efficiency
was observed for the incorporation of dM7AITP opposite the
ICS analogues by 10-20-fold relative to the incorporation of
d7AITP. The effect of the C6-methyl group was even larger
for the incorporation of the unnatural triphosphate opposite
native templates, where none of the rates exceeded 104 M-1
min-1 (Table 7).
3.1.3. Electronic Effects. Electronic effects may also play
an important role in the stability of unnatural base pairs in
DNA.34-36 The 7AI ring has a single nitrogen atom (N7) and
is otherwise hydrophobic. Aza-substitution is expected to affect
the physical properties of the nucleobases. First, the presence
of the extra nitrogen atoms in the polyaza heterocycles decreases
the basicity at N7, relative to that of 7AI, due to the inductive
electron-withdrawing effect of the added nitrogen atoms and
to unfavorable charge localization in the protonated forms
3.1.2. Effect of Propynyl Substitution. Previously, with
isocarbostyril base pairs, it was observed that unsymmetrical
(28) Morales, J. C.; Kool, E. T. J. Am. Chem. Soc. 2000, 122, 1001-
1007.
(29) Moran, S.; Ren, R. X.-F.; Kool, E. T. Proc. Natl. Acad. Sci. U.S.A.
1997, 94, 10506-10511.
(34) Bergman, E. D.; Weiler-Feilchenfeld. In The Dipole Moments of
Purines; Bergman, E. D., Pullman, B., Eds.; The Israel Academy of Sciences
and Humanities: Jerusalem, 1971; pp 21-28.
(30) Moran, S.; Ren, R. X.-F.; Rumney, S. I.; Kool, E. T. J. Am. Chem.
Soc. 1997, 119, 2056-2057.
(35) Lister, J. H. The Purines. Supplement 1; John Wiley and Sons: New
York, 1996; Vol. 54.
(31) Goodman, M. F. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 10493-
10495.
(32) Morales, J. C.; Kool, E. T. Nature Struct. Biol. 1998, 5, 950-954.
(33) Kool, E. T. Biopolymers 1998, 48, 3-17.
(36) Hurst, D. T. An Introduction to the Chemistry and Biochemistry of
Pyrimidines, Purines and Pteridines; Page Bros.: Norwich, Great Britain,
1980.

Optimization of Interbase Hydrophobic Interactions
J. Am. Chem. Soc., Vol. 122, No. 32, 2000 7629
relative to the unprotonated forms of these heterocycles.36 For
example, the pKa of pyrazine (pKa ) 0.7) is significantly lower
than that of pyridine (pKa ) 5.2), as is the N3 pKa of
2′-deoxyxanthosine (pKa ) 5.7) relative to that of 2′-deoxy-7-
deazaxanthosine (pKa ) 7.2).2,37 Second, aza-substitution is also
expected to affect the dipole moment of the base.34,36 In analogy
to native purine rings, the effects of aza-substitution on the
overall electron distribution in the 7AI analogues may be
expected to result from the sharing of electron density between
nitrogen atoms of the fused five- and six-membered rings.34,36
The N4-aza-substitution of PP should result in a more π-electron
deficient pyrazine ring and should contribute to a dipole moment
oriented toward the six-membered ring, more similar to the
native purine ring dipole moment. The N3-aza-substitution of
ImPy is expected to result in a more π-electron deficient pyrrole
ring and should contribute to a dipole oriented toward the five-
membered ring, opposite to the case with a purine ring system.
Finally, aza-substitution is also expected to affect the polariz-
ability of the base. A significant component of base stacking
results from polarization forces, which manifest themselves
structurally as the partial overlap of the polar bonds of one
nucleobase with the polarizable ring system of an adjacent
base.38 The addition of heteroatoms to otherwise nonpolar
hydrophobic rings, such as benzene and naphthalene, has a
pronounced effect on the structure of these molecules.38 The
nonpolar molecules form structures with the characteristic
herringbone pattern with no ring stacking, while those bearing
heteroatom substituents crystallize in forms dominated by ring-
stacking interactions with the polar heteroatoms packing with
the polarizable ring system of an adjacent molecule.38
The Tm data in Table 1 reveal that both N3 of ImPy and N4
of PP result in a consistent decrease in duplex stability. The
destabilization is unlikely to result from direct interactions
between pairing bases due to the large backbone deformations
that would be necessary to position the nitrogen atoms in a
region of the duplex accessible to the pairing base. This is
consistent with preliminary NMR data which indicate that the
7AI:ICS packs in DNA without significant backbone deforma-
tions or intercalation. Since the extent of destabilization is
roughly independent of the pairing base, it may derive from
less than optimal stacking interactions. However, PP and ImPy
are expected to stack more favorably with adjacent bases due
to the predicted overlap with polarizable regions of the adjacent
bases. Moreover, poor stacking would also be surprising, at least
for ImPy, considering its more purine-like imidizo ring structure.
It seems more likely that the decrease in duplex stability may
result from a decrease in the stability of H-bonding interactions
with the ordered water molecules along the minor groove39-43
due to the decreased ability of the minor groove N7 of 7AI to
accept a H-bond relative to the corresponding minor groove
nitrogen atom of guanine or adenine. The H-bond-accepting
ability of the minor groove N7 of PP and ImPy is further
compromised by the inductive electron-withdrawing effects of
aza-substitution. Despite these effects, the overall reduction in
duplex stability is not a significant obstacle to the use of these
unnatural bases. The stability of the unnatural base pairs
comprised of PP and ImPy is comparable, or only slightly
reduced, relative to that of native base pairs.
The effects of nitrogen substitution were also examined in
the context of KF-mediated DNA synthesis. The effect on
unnatural base pair synthesis was negligible for modification
of the templating base with either N3 or N4 substitution. In the
template, 7AI, PP, and ImPy directed the incorporation of each
ICS analogue triphosphate with virtually identical rates. How-
ever, unnatural base pair synthesis was more affected when these
substitutions were made in the triphosphate. Compared to the
insertion of d7AITP opposite the ICS analogues, dImPyTP was
inserted less efficiently (by 2-7-fold), resulting predominately
from a decrease in apparent kcat, while dPPTP was inserted more
efficiently (by 2-8-fold), resulting primarily from a decrease
in apparent KM(triphosphate). The largest difference observed
was in the insertion of dPPTP opposite 5MICS (14-fold smaller
apparent KM) or MICS (7-fold smaller apparent KM, and 8-fold
increased kcat/KM). It seems unlikely that the differing behavior
of the two aza-substituted triphosphates results from differing
bascisity of N7, as each analogue is less basic. The opposite
effects that N3- and N4-aza-substitution have on KF recognition
imply a role for the dipole moment of the bases, as the addition
of the electron-withdrawing heteroatoms should contribute to
dipole moments oriented in opposite directions. Unnatural
nucleobases that are functionalized with heteroatoms at a variety
of positions are currently being further examined.
Unlike hydrophobic pair synthesis, the rates with which KF
inserted native bases opposite the template were sensitive to
aza-substitution. Generally, the native triphosphates were in-
serted by KF more efficiently opposite both ImPy and PP, as
opposed to 7AI, predominately due to an increase in apparent
kcat. The effect is most dramatic for the insertion of dCTP
opposite both ImPy and PP templates, where the apparent kcat
for insertion of dCTP was increased 15-fold, relative to the
insertion of dCTP opposite 7AI. When present as the triphos-
phate, the two N-substitutions had different effects on KF
recognition. Both dImPyTP and dPPTP bound to the enzyme-
template complex most tightly opposite dC. However, the only
consistent and significant effect was tighter binding of dPPTP
opposite native templates, with KM(dPPTP) 3-9-fold smaller
than KM(d7AITP). The effect was greatest opposite dC in the
template, which when combined with a 3-fold increase in
insertion efficiency results in a greater than 20-fold increase in
the pairing of dPPTP opposite dC, relative to that of d7AITP.
The electronic effects resulting from C3- or C4-aza-substitution
selectively favored pairing with deoxycytosine. Like the efficient
insertion of dCTP opposite P7AI discussed above, the efficient
pairing of PP and ImPy with deoxycytosine is not reflected in
the thermal stability of the base pair, as dC:PP and dC:ImPy
are both 2.2 °C less stable than dC:7AI, again implying that
the factors governing efficient replication are decoupled from
those governing pair stability in duplex DNA. The specific
increase in efficiency must result from either polymerase-
mediated interactions or an early insertion transition state bearing
little resemblance to products.
(37) Gilchrist, T. L. Heterocyclic Chemistry; Longman Scientific &
Technical: Essex, 1985.
(38) Bugg, C. E. In Solid-state packing patterns of purine bases;
Bergmann, E. D., Pullman, B., Eds.; The Israel Academy of Sciences and
Humanities: Jerusalem, 1971; pp 178-204.
(39) Drew, H. R.; Dickerson, R. E. J. Mol. Biol. 1981, 151, 535-56.
(40) Drew, H. R.; Travers, A. A. Cell 1988, 37.
(41) Kopka, M. L.; Fratini, A. V.; Drew, H. R.; Dickerson, R. E. J. Mol.
Biol. 1983, 163, 129-46.
(42) Liepinsh, E.; Otting, G.; Wu¨thrich, K. Nucleic Acids Res. 1992,
20, 6549-53.
(43) Kubinec, M. G.; Wemmer, D. E. J. Am. Chem. Soc. 1992, 114,
8739-8740.
3.2. Optimization of Interbase Hydrophobic Interactions
for Efficient Replication. The efficiencies of unnatural base
insertion opposite another unnatural base in the template show
a surprising trend: while modifications of the incoming triph-
osphate base may have large effects on the rate of base pair
synthesis, the base in the template is of less importance. Each
triphosphate is inserted with little variation in rate opposite each

7630 J. Am. Chem. Soc., Vol. 122, No. 32, 2000
Wu et al.
unnatural base in the template. Opposite each 7AI analogue in
the template dMICSTP is inserted the most efficiently, followed
by dPICSTP and dICSTP, and finally d5MICSTP. Opposite each
ICS analogue in the template, dP7AITP is inserted the most
efficiently, followed by dPPTP, d7AITP, dImPyTP, and
dM7AITP. The effects were the greatest for the insertion of
the 7AI analogue triphosphates opposite the ICS analogues in
the template. The insertion of a given 7AI analogue triphophate
varied by 2-7-fold, depending on the unnatural base in the
template, while the insertion of the 7AI analogue triphosphates
opposite a given template varied by 200-600-fold. Relative to
the unnatural base in the template, the unnatural base of the
triphosphate was 2-3 orders of magnitude more sensitive to
the addition of methyl groups in the developing major or minor
groove, propynyl substitution, or isostructural aza-substitution.
4. Conclusion and Future Prospects
Hydrophobicity is a strong and selective force in biological
systems. The hybridization and substrate properties of hydro-
phobic bases may be tuned by judicious electronic and sub-
stituent modification. We have derivatized the earlier reported
7AI:ICS base pair and characterized the resulting PP:MICS base
pair. PP:MICS is the first unnatural base pair reported that is
efficiently accepted by a DNA polymerase with kinetic selectiv-
ity against all possible mispairs. We are further examining
derivatives of these hydrophobic ring structures, as well as
others, in a continuing effort to optimize the interbase hydro-
phobic interactions required for information storage and re-
trieval.
5. Experimental Section
This asymmetry is not easily explained by any simple model
which relies on interbase interactions for correct base-pairing.
For example, improvements in shape complementarity due to
methyl group substitution should stabilize the nascant base pair,
regardless of whether the modified base is in the template or
the triphospahte. This asymmetry also has important implications
for our efforts to develop a third orthogonal base pair for
expansion of the genetic alphabet. While rates for unnatural base
pair synthesis are sensitive to base modification, and may
approach those characteristic of native DNA synthesis, special
care must be taken to prevent the efficient mispairing of the
hydrophobic bases.
General Experimental Methods. NMR spectra were obtained from
Bruker ARX400, ARX500, and ARX600 spectrometers. High-resolution
mass spectra were obtained by the Mass Spectroscopy Facilities at
TSRI. Solvents and reagents were used as supplied from commercial
sources with the following exceptions: all moisture-sensitive reagents
were either distilled prior to use or stored over 4-Å molecular sieves
for greater than 12 h. Tetrahydrofuran was distilled from sodium
benzophenone ketyl immediately prior to use. Dichloromethane was
distilled from calcium hydride. All reactions involving moisture-
sensitive reagents were performed under an argon atmosphere.
General Toluoyl Deprotection Procedure. To a stirred solution
of nucleoside (1 equiv) in CH3OH (0.1 M) at room temperature was
added NaOMe (>2.2 equiv). After the reaction was complete, the
reaction was quenched via addition of solid NH4Cl (excess) and
concentrated. Purification via column chromatography on silica gel
(gradient of CH3OH in CH2Cl2) afforded the nucleoside.
General Triphosphate Synthesis Procedure. Proton sponge (1.5
equiv) and nucleoside (1 equiv) were dissolved in trimethyl phosphate
(final concentration ∼0.3 M) and cooled to 0 °C. POCl3 (1.05 equiv)
was added dropwise, and the lavender slurry was stirred at 0 °C for an
additional 2 h. Tributylamine (4 equiv) was added, followed by a
solution of tributylammonium pyrophosphate (>2.5 equiv) in DMF (to
final concentration ∼0.15 M). After 1 min, the reaction was quenched
by addition of 1 M aqueous triethylammonium bicarbonate (20 vol
equiv). The resulting crude solution was lyophilized and purified by
reverse-phase (C18) HPLC (4-35% CH3CN in 0.1 M NEt3-HCO3,
pH 7.5) to afford the triphosphate as a white solid.
General Phosphoramidite Synthesis Procedure. To a solution of
nucleoside (1 equiv) in pyridine (0.2 M) was added triethylamine (3
equiv), followed by DMTr-Cl (1.5 equiv). After being stirred for 30
min at room temperature, the reaction mixture was concentrated.
Purification via column chromatography on silica gel (50-100% ethyl
acetate in hexane) afforded the tritylated product, which was dissolved
in CH2Cl2 (0.1 M) and cooled to 0 °C. A catalytic amount of DMAP
was added, followed by triethylamine (5 equiv) and 2-cyanoethyl
diisopropylaminochlorophosphoramidite (2 equiv). After 15 min, the
reaction mixture was partitioned between CH2Cl2 and saturated aqueous
NaHCO3. The layers were separated, and the aqueous layer was
extracted twice with CH2Cl2. The combined organics were dried over
Na2SO4, filtered, and concentrated. Purification via column chroma-
tography on silica gel (15-50% ethyl acetate in 5% triethylamine/
hexane) afforded the desired phosphoramidite as a mixture of two
diastereomers.
General Procedure for Oligonucleotide Synthesis and Purifica-
tion. Oligonucleotides containing unnatural bases were synthesized with
an ABI 392 DNA/RNA synthesizer using standard â-cyanoethyl
phosphoramidite chemistry. Oligonucleotides were cleaved from solid
support and deprotected by 12 h of incubation with concentrated
ammonia at 55 °C. Oligonucleotides were then purified by preparative
15% denaturing polyacrylamide gel electrophoresis, isolated from the
gel, and quantitated by absorbance at 260 nm.
3.3. An Unnatural Base Pair with Improved Kinetic
Properties. Efforts to expand the genetic code are reliant on
development of a third base pair to supplement the naturally
existing base pairs, dA:dT and dG:dC. The nucleobases that
comprise the third base pair must be thermodynamically and
kinetically orthogonal to the natural bases. Base pairs reported
above between 7AI analogues and ICS analogues are significant
improvements over those previously reported.18,19 For example,
the reasonable stability of the M7AI:5MICS pair, as well as
the improved selectivity against self-pair synthesis, make it a
potentially interesting target for further optimization. However,
the low efficiency for M7AI:5MICS synthesis by KF currently
limits its practical utility.
The PP:MICS unnatural base pair is more promising, even
though it is not thermodynamically selective against the MICS:
MICS self-pair. In the template, PP directs KF to incorporate
dMICSTP very efficiently (kcat/KM ) 2.0 × 106 M-1 min-1),
while dPPTP is inserted opposite MICS with an equally efficient
kcat/KM value of 1.4 × 106 M-1 min-1. These rates are both
only approximately 20-fold reduced relative to those typical of
native synthesis. The PP:MICS unnatural pair is also synthesized
4- and 12-fold faster than the self-pairs (1.7 × 105 and 3.3 ×
105 M-1 min-1 for PP:PP and MICS:MICS self-pairs, respec-
tively). The most efficiently synthesized mispairs between
unnatural and native bases are dCTP insertion opposite PP (9.3
× 104 M-1 min-1) and dATP insertion opposite MICS (2.4 ×
104 M-1 min-1), resulting in selectivities of 21- and 58-fold
favoring correct unnatural pair synthesis. Moreover, preliminary
results show that the PP:MICS pair is also extended by KF with
reasonable efficiency.
While these rates are impressive, the selectivity is several
orders of magnitude reduced relative to what will be required
for information storage. However, we emphasize that the PP:
MICS base pair is the first unnatural base pair to be incorporated
into DNA with a measurable selectivity against all possible
mispairs.
General Polymerase Kinetic Assay Protocol. Primer was 5′-end
32P-labeled with T4 polynucleotide kinase and [γ -32P]ATP (10 nCi/10
µL reaction). Primer-template duplexes were annealed by mixing in

Optimization of Interbase Hydrophobic Interactions
J. Am. Chem. Soc., Vol. 122, No. 32, 2000 7631
the reaction buffer, heating to 90 °C, and slowly cooling to room
temperature. Assay conditions: 40 nM template-primer duplex, 0.11-
1.34 nM enzyme, 50 mM Tris buffer (pH 7.5), 10 mM MgCl2, 1 mM
DTT, and 50 µg/mL BSA. The reactions were initiated by adding the
DNA-enzyme mixture to an equal volume (5 µL) of a 2× dNTP stock
solution, incubated at room temperature for 1-10 min, and quenched
by the addition of 20 µL of loading buffer (95% formamide, 20 mM
EDTA). The reaction mixture (5 µL) was then analyzed by 15%
polyacrylamide gel electrophoresis. Radioactivity was quantified using
a PhosphorImager (Molecular Dynamics) and the ImageQuant program.
The kinetic data were fit to the Michaelis-Menten equation using the
program Kaleidograph (Synergy Software). Data presented are averages
of duplicates or triplicates.
aqueous 5% EDTA and brine, dried over Na2SO4, filtered, and
concentrated. Purification by column chromatography on silica gel (20%
ethyl acetate in hexane) afforded 13 (330 mg). To a stirred solution of
13 (330 mg, 0.65 mmol) in CH3OH (10 mL) was added 1 M NaOMe
(2 mL). After 45 min, the excess NaOMe was quenched with NH4Cl
(∼100 mg). The resulting slurry was concentrated and purified by
column chromatography on silica gel (hexane/ethyl acetate/MeOH 4:4:
1), which afforded 4 (160 mg, 59% over three steps): 1H NMR (400
MHz, CD3OD) δ 8.25 (1H, d, J ) 4.3 Hz), 8.04 (1H, dd, J ) 8.1, 1.5
Hz), 7.35 (1H, s), 7.15 (1H, dd, J ) 8.1, 4.8 Hz), 6.28 (1H, dd, J )
9.5, 5.7 Hz), 4.75 (1H, d, J ) 5.1 Hz), 4.21 (1H, m), 3.96 (1H, m),
3.80 (1H, m), 3.18 (1H, ddd), 2.23 (1H, ddd, J ) 13.5, 5.9, 3.7 Hz),
2.10 (3H, s); HRMS calcd for C15H17N2O5 (MH+) 273.1234, found
273.1235.
Compound 1. To a stirred solution of 6-methyl-7-azaindole (56 mg,
0.427 mmol) in DMF (2 mL) at 0 °C was added sodium hydride (12
mg, 0.512 mmol). The resulting dark brown mixture was stirred at 0
°C for 15 min, at which time it was added slowly dropwise to a solution
of chloroglycoside (199 mg, 0.512 mmol) in DMF (3 mL). After being
stirred at 0 °C for 1 h, the reaction mixture was partitioned between
saturated aqueous NaHCO3 (15 mL) and ethyl acetate (20 mL). The
layers were separated, and the aqueous layer was extracted with 2 ×
20 mL of ethyl acetate. The combined organics were dried over Na2-
SO4, filtered, and concentrated. Purification via column chromatography
on silica gel (25-70% ethyl acetate in hexane) afforded bis-tolyl
nucleoside 10 (105 mg, 51% yield), which was dissolved in CH3OH
(4 mL) and deprotected using the general procedure outlined above.
Purification by column chromatography on silica gel (1-4% CH3OH
in CH2Cl2) afforded nucleoside 1 (31 mg, 58%): 1H NMR (600 MHz,
CDCl3) δ 7.82 (1H, d, J ) 8.0 Hz), 7.13 (1H, d, J ) 3.6 Hz), 6.96
(1H, d, J ) 8.0 Hz), 6.37 (1H, m), 6.29 (1H, dd, J ) 9.8, 5.6 Hz), 4.79
(1H, d, J ) 5.2 Hz), 4.21 (1H, s), 4.00 (1H, dd, J ) 12.5, 1.7 Hz),
3.84 (1H, dd, J ) 12.5, 1.6 Hz), 3.25 (1H, ddd, J ) 13.6, 9.8, 5.2 Hz),
2.61 (3H, s), 2.21 (1H, dd, J ) 13.5, 5.6 Hz); 13C NMR (150 MHz,
CDCl3) δ 151.4, 145.6, 130.4, 130.0, 129.1, 127.9, 121.2, 116.8, 100.2,
90.2, 88.7, 73.9, 63.7, 39.9, 23.4; HRMS calcd for C13H17N2O3 (MH+)
249.1239, found 249.1232.
Compound 2. Compound 2 was synthesized according to a literature
procedure:44 1H NMR (400 MHz, CD3OD) δ 8.61 (1H, s), 8.36 (1H,
dd, J ) 4.9, 1.4 Hz), 8.10 (1H, dd, J ) 8.1, 1.4 Hz), 7.36 (1H, dd, J
) 8.1, 4.8 Hz), 6.57 (1H, dd, J ) 7.9, 6.0 Hz), 4.61 (1H, m), 4.09
(1H, dd, J ) 5.9, 3.1 Hz), 3.85 (1H, dd, J ) 12.2, 3.1 Hz), 3.75 (1H,
dd, J ) 12.2, 3.4 Hz), 2.89 (1H, ddd, J ) 13.6, 7.9, 5.9 Hz), 2.43 (1H,
ddd, J ) 13.4, 6.1, 2.8 Hz); 13C NMR (100 MHz, CD3OD) δ 147.0,
145.3, 145.1, 137.1, 129.2, 120.2, 89.8, 87.1, 73.1, 63.7, 41.3.
Compound 3: 1H NMR (400 MHz, CD3OD) δ 8.37 (1H, d, J )
2.7 Hz), 8.24 (1H, d, J ) 2.7 Hz), 8.05 (1H, d, J ) 3.8 Hz), 6.68 (1H,
dd, J ) 8.1, 6.0 Hz), 6.67 (1H, d, J ) 3.8 Hz), 4.53 (1H, m), 3.99 (1H,
dd, J ) 6.6, 4.1 Hz), 3.77 (1H, dd, J ) 12.0, 3.5 Hz), 3.70 (1H, dd, J
) 12.0, 4.1 Hz), 2.72 (1H, ddd, J ) 13.8, 7.7, 6.0 Hz), 2.35 (1H, ddd,
J ) 13.5, 6.2, 3.0 Hz); HRMS calcd for C11H14N3O3 (MH+) 236.1035,
found 236.1034.
Compound 4. To a stirred solution of 12 (450 mg, 0.96 mmol) in
CH2Cl2 (5 mL) was added 1 M ICl in CH2Cl2 (1.3 mL, 1.3 mmol), and
the mixture was heated to 50 °C for 1 min. After cooling to room
temperature over 30 min, the mixture was diluted with CH2Cl2 (20
mL), and the excess ICl was quenched with saturated aqueous Na2S2O3
(15 mL). The layers were separated, and the aqueous layer was extracted
with 3 × 20 mL of CH2Cl2. The combined organic layers were washed
with brine, dried over Na2SO4, filtered, and concentrated in vacuo.
Purification by column chromatography on silica gel (20% ethyl acetate
in hexane) afforded 540 mg of white crystals, which were suspended
in NEt3 (20 mL) in a pressure tube. Argon was bubbled through the
mixture for 15 min, and (Ph3P)2PdCl2 (63 mg, 0.09 mmol) and CuI
(34 mg, 0.18 mmol) were added. The mixture was cooled to -78 °C,
and propyne (∼1 mL) was condensed in the tube. The reaction was
sealed and allowed to warm to room temperature over 4 h. Prior to
quenching, the reaction mixture was vented at -78 °C and then warmed
to room temperature and concentrated. The crude residue was dissolved
in CHCl3 (50 mL), and the organic layer was washed two times with
Compound 5. NaH (60%, 70 mg, 1.7 mmol) was suspended in
acetonitile (3 mL) and cooled to 0 °C. Compound 14 (265 mg, 1.65
mmol) was added in acetonitile (2 mL) over 15 min. The cooling bath
was removed, and the mixture was stirred for 1 h. Chloroglycoside 7
(622 mg, 1.6 mmol) was added in four portions, and the mixture was
stirred for 2 h. The slurry was filtered through Celite, and the residue
was washed with Et2O (∼20 mL). The filtrate was concentrated, and
purification by column chromatography on silica gel afforded 560 mg
of a 1:1 mixture of the N-glycosidic and the O-glycosidic compounds.
To a solution of the mixture in CH3OH (10 mL) and THF (2 mL) was
added 1 M NaOMe (3 mL). After 45 min, the excess NaOMe was
quenched with solid NH4Cl (∼200 mg). The resulting slurry was
concentrated and purified by column chromatography on silica gel
(hexane/ethyl acetate/MeOH 4:4:1), which afforded 160 mg of 5 (34%
over two steps): 1H NMR (250 MHz, CDCl3) δ 7.44 (1H, d, J ) 7.6,
7.3 Hz), 7.31 (1H, d, J ) 7.3 Hz), 7.28 (1H, d, J ) 7.6 Hz), 7.19 (1H,
d, J ) 7.5 Hz), 6.50 (1H, dd, J ) 7.2, 6.3 Hz), 6.43 (1H, d, J ) 7.5
Hz), 4.60 (1H, ddd, J ) 6.5, 4.0, 1.0 Hz), 4.10 (1H, ddd, J ) 6.5, 4.0,
1.0 Hz), 3.94 (1H, dd, J ) 12.1, 3.3 Hz), 3.85 (1H, dd, J ) 12.1, 4.0
Hz), 2.92 (3H, s), 2.48 (1H, ddd, J ) 13.5, 6.3, 4.0 Hz), 2.37 (1H,
ddd, J ) 13.5, 7.2, 1.0 Hz); HRMS calcd for C15H17NO4Na (MNa+)
298.1050, found 298.1050.
Compound 6: 1H NMR (400 MHz, CD3OD) δ 8.17 (1H, d, J )
8.2 Hz), 7.72 (1H, d, J ) 7.6 Hz), 7.40 (1H, s), 7.34 (1H, dd, J ) 8.4,
1.5 Hz), 6.68 (1H, dd, J ) 7.3, 6.2 Hz), 6.64 (1H, d, J ) 7.6 Hz), 4.42
(1H, dt, J ) 6.5, 3.2 Hz), 3.99 (1H, dd, J ) 7.5, 3.8 Hz), 3.82 (1H, dd,
J ) 12.0, 3.5 Hz), 3.76 (1H, dd, J ) 11.9, 4.4 Hz), 3.30 (1H, m), 2.47
(3H, s), 2.40 (1H, ddd, J ) 13.5, 6.2, 3.5 Hz), 2.18 (1H, ddd, J )
13.6, 7.3, 6.5 Hz); 13C NMR (150 MHz, CD3OD) δ 158.0, 139.4, 133.2,
124.1, 122.7, 122.4, 121.4, 118.7, 102.4, 83.4, 80.8, 66.9, 57.5, 36.5,
16.2; HRMS calcd for C15H17NO4Na (MNa+) 298.1050, found 298.1062.
Compound 11. To a stirred solution of 9 (100 mg, 0.840 mmol) in
CH3CN (4.5 mL) was added NaH (36 mg, 0.900 mmol, 60% dispersion
in mineral oil), and the resulting mixture was stirred at room temperature
for 10 min. The sodium salt was added to chloroglycoside 7 (400 mg,
1.028 mmol) and stirred at room temperature for 10 min, at which
time the reaction was partitioned between ethyl acetate (25 mL) and
saturated aqueous NaHCO3 (20 mL). The layers were separated, and
the aqueous layer was extracted with 2 × 20 mL of ethyl acetate. The
combined organics were dried over Na2SO4, filtered, and concentrated.
Purification via column chromatography on silica gel (15-30% ethyl
acetate in hexane) afforded the bis-protected nucleoside 11 (161 mg,
41%): 1H NMR (400 MHz, CDCl3) δ 8.38 (1H, m), 8.08 (2H, d, J )
6.4 Hz), 8.02 (2H, d, J ) 6.5 Hz), 7.78 (1H, d, J ) 2.1 Hz), 7.36 (2H,
d, J ) 6.4 Hz), 7.34 (1H, s), 7.33 (2H, d, J ) 6.7 Hz), 6.91 (1H, dd,
J ) 6.9, 4.4 Hz), 6.83 (1H, m), 5.87 (1H, m), 4.80 (1H, dd, J ) 9.7,
3.2 Hz), 4.73 (1H, dd, J ) 9.4, 3.2 Hz), 4.69 (1H, m), 3.08 (1H, ddd,
J ) 11.6, 6.6, 5.0 Hz), 2.83 (1H, ddd, J ) 11.4, 4.5, 1.8 Hz), 2.52
(3H, s), 2.50 (3H, s).
Compound 14. Data are presented for comparison to literature
values:45 1H NMR (500 MHz, CDCl3) δ 7.48 (1H, dd, J ) 7.9, 7.3
Hz), 7.35 (1H, d, J ) 7.9 Hz), 7.22 (1H, d, J ) 7.3 Hz), 7.01 (1H, d,
J ) 7.0 Hz), 6.44 (1H, d, J ) 7.0 Hz), 2.93 (3H, s); 13C NMR (125
MHz) δ 221.7, 164.5, 141.9, 131.8, 129.8, 127.1, 124.5, 106.9, 23.7.
(45) Hirao, K.; Tsuchiya, R.; Yano, Y.; Tsue, H. Heterocycles 1996,
42, 415-422.
(44) Wenzel, T.; Seela, F. HelV. Chim. Acta 1996, 79, 169-178.

7632 J. Am. Chem. Soc., Vol. 122, No. 32, 2000
Wu et al.
Compound 17. To a slurry of 15 (800 mg, 5.0 mmol) in CH3CN
(14.5 mL) was added bis(trimethylsilyl)acetamide (1.24 mL, 5.0 mmol).
The mixture became homogeneous over 30 min, at which time it was
added to chloroglycoside 7 (1.5 g, 3.86 mmol) in CH3CN (17.4 mL).
The resulting slurry was cooled to 0 °C, and SnCl4 (85 µL, 0.73 mmol)
was added. After 30 min, the reaction was partitioned between ethyl
acetate (100 mL) and saturated aqueous NaHCO3 (100 mL). The layers
were separated, and the aqueous layer was extracted with 2 × 100 mL
of ethyl acetate. The combined organics were dried over Na2SO4,
filtered, and concentrated. Purification via column chromatography on
silica gel (15-40% ethyl acetate in hexane) afforded protected
nucleoside 17 (253 mg, 13%): 1H NMR (500 MHz, CDCl3) δ 8.38
(1H, d, J ) 8.2 Hz), 8.06 (2H, d, J ) 8.4 Hz), 8.01 (2H, d, J ) 8.4
Hz), 7.49 (1H, d, J ) 7.7 Hz), 7.29-7.40 (6H, m), 6.95 (1H, dd, J )
8.3, 5.5 Hz), 6.45 (1H, d, J ) 7.7 Hz), 5.72 (1H, m), 4.81 (2H, m),
4.67 (1H, dd, J ) 6.1, 3.4 Hz), 2.94 (1H, ddd, J ) 14.3, 5.4, 1.8 Hz),
2.55 (3H, s), 2.52 (3H, s), 2.49 (3H, s), 2.42 (1H, m); HRMS calcd for
C31H29NO6Na (MNa+) 534.1893, found 534.1897.
Compound 25: 1H NMR (400 MHz, CDCl3) δ 8.32 (1H, dd, J )
4.8, 1.4 Hz), 8.23 (1H, d, J ) 8.7 Hz), 8.05 (1H, dt, J ) 8.0, 1.4 Hz),
7.39 (2H, m), 7.15-7.30 (9H, m), 6.76 (4H, m), 6.56 (1H, m) 4.77
(1H, m), 4.29 (1H, m), 3.55-3.90 (10H, m), 3.30-3.40 (2H, m), 2.92
(1H, m), 2.64 (1H, m), 2.61 (1H, t, J ) 6.4 Hz), 2.46 (1H, t, J ) 6.4
Hz), 1.16-1.19 (9H, m), 1.11 (3H, d, J ) 6.8 Hz); 31P NMR (140
MHz, CDCl3) δ 149.3, 149.2.
1
Compound 27: H NMR (500 MHz, CDCl3) δ 8.31 (1H, m), 7.97
(1H, d, J ) 7.7 Hz), 7.56 (0.5H, s), 7.53 (0.5H, s), 7.42-7.45 (2H,
m), 7.31-7.34 (4H, m), 7.18-7.29 (3H, m), 7.13 (1H, m), 6.76-6.85
(4H, m), 4.69 (1H, m), 4.18-4.25 (1H, m), 3.79 (3H, s), 3.78 (3H, s),
3.54-3.70 (2H, m), 3.36 (0.5H, dd, J ) 9.9, 4.0 Hz), 3.25-3.32 (1.5H,
m), 2.57-2.64 (2H, m), 2.47-2.53 (1H, m), 2.45 (1H, t, J ) 6.2 Hz),
2.14 (3H, s), 1.19 (3H, d, J ) 6.7 Hz), 1.18 (3H, d, J ) 6.4 Hz), 1.16
(3H, d, J ) 6.3 Hz),1.09 (3H, d, J ) 7.0 Hz).
1
Compound 28: H NMR (500 MHz, CDCl3) δ 7.69 (0.5H, d, J )
7.3 Hz), 7.60 (0.5H, d, J ) 7.5 Hz), 7.43-7.48 (3H, m), 7.19-7.37
(9H, m), 6.84 (2H, d, J ) 8.5 Hz), 6.82 (2H, d, J ) 8. 8 Hz), 6.70
(0.5H, dd, J ) 6.3, 6.2 Hz), 6.67 (0.5H, dd, J ) 6.3, 6.2 Hz), 6.26
(0.5H, d, J ) 7.5 Hz), 6.25 (0.5H, d, J ) 7.5 Hz), 4.65 (0.5H, m), 4.60
(0.5H, m), 4.19 (1H, m), 3.79 (3H, s), 3.78 (3H, s), 3.48-3.61 (4H,
m), 3.32-3.38 (1H, m), 2.92 (3H, s), 2.68 (0.5H, ddd, J ) 13.5, 6.2,
3.7 Hz), 2.62 (0.5H, ddd, J ) 13.5, 6.2, 3.7 Hz), 2.61 (1H, dd, J )
6.4, 6.2 Hz), 2.40 (1H, dd, J ) 6.6, 6.4 Hz), 2.26 (0.5H, ddd, J )
13.5, 6.8, 6.3 Hz), 2.24 (0.5H, ddd, J ) 13.5, 6.8, 6.3 Hz), 2.06 (3H,
s), 1.17 (3H, d, J ) 6.6 Hz), 1.16 (3H, d, J ) 6.8), 1.15 (3H, d, J )
6.7 Hz), 1.05 (3H, d, J ) 6.8 Hz).
Compound 18: 31P NMR (140 MHz, 50 mM Tris, 2 mM EDTA,
pH 7.5 in D2O) δ -6.03 (br), -10.41 (d, J ) 15.4 Hz), -21.54 (br).
Compound 19: 31P NMR (140 MHz, 50 mM Tris, 2 mM EDTA,
pH 7.5 in D2O) δ -6.35 (d, J ) 16.8 Hz), -10.51 (d, J ) 16.8 Hz),
-21.68 (t, J ) 16.8 Hz).
Compound 20: 31P NMR (140 MHz, 50 mM Tris, 2 mM EDTA,
pH 7.5 in D2O) δ -5.16 (d, J ) 18.0 Hz), -9.94 (d, J ) 16.8 Hz),
-21.27 (dd, J ) 18.0, 17.4 Hz).
Compound 21: 31P NMR (140 MHz, 50 mM Tris, 2 mM EDTA,
pH 7.5 in D2O) δ -5.68 (d, J ) 20.9 Hz), -10.46 (d, J ) 20.1 Hz),
-21.84 (dd, J ) 20.9, 20.1 Hz).
Compound 22: 31P NMR (140 MHz, 50 mM Tris, 2 mM EDTA,
pH 7.5 in D2O) δ -5.82 (d, J ) 21.4 Hz), -10.52 (d, J ) 19.8 Hz),
-21.90 (t, J ) 20.6 Hz).
Acknowledgment. Funding was provided by the National
Institutes of Health (GM 60005 to F.E.R.), the Skaggs Institute
for Chemical Biology (F.E.R. and P.G.S.), and a National
Institutes of Health postdoctoral fellowship (F32 GM19833-01
to A.K.O.).
Compound 23: 31P NMR (140 MHz, 50 mM Tris, 2 mM EDTA,
pH 7.5 in D2O) δ -5.79 (d, J ) 18.5 Hz), -10.68 (d, J ) 17.2 Hz),
-21.97 (dd, J ) 18.5, 17.2 Hz).
Compound 24: 1H NMR (400 MHz, CDCl3) δ 7.74 (1H, d, J )
7.9 Hz), 7.43 (2H, m), 7.32 (5H, m), 7.17-7.25 (3H, m), 6.93 (1H, d,
J ) 7.9 Hz), 6.88 (1H, m), 6.77 (4H, m), 6.38 (1H, dd, J ) 3.7, 1.9
Hz), 4.75 (1H, m), 4.21 (1H, m), 3.57-3.89 (10H, m), 3.36 (1H, m),
3.28 (1H, m), 2.71 (1H, m), 2.62 (1H, t, J ) 2.4 Hz), 2.58 (3H, d, J )
1.7 Hz), 2.54 (1H, m), 2.45 (1H, m), 1.16-1.20 (9H, m), 1.09 (3H, d,
J ) 6.8 Hz); 31P NMR (140 MHz, CDCl3) δ 149.1, 148.9.
Supporting Information Available: Experimental proce-
dures and characterizations (PDF). This material is available
free of charge via the Internet at http://pubs.acs.org.
JA0009931