Angewandte
Chemie
Based on the efficiency and fidelity of 2Py self-pair
extension, we synthesized the corresponding nucleoside
triphosphate (see the Supporting Information)and examined
the efficiency of Kf-mediated self-pair synthesis (Table 3).
to its efficient synthesis. dCTP was not efficiently inserted
opposite any of the pyridyl analogues. Overall, the rates at
which 2Py directs the synthesis of mispairs are similar to those
of BEN,[3c] a result suggesting that the minor-groove H-bond
acceptor has only a minor effect on mispair synthesis.
The data demonstrate that the phenyl-ring nucleobase
scaffold may be optimized for both base-pair stability and
replication by derivatization with an ortho nitrogen atom.
This H-bond acceptor, which is at the a position relative to
the glycosidic linkage, appears to be well positioned to
mediate critical minor-groove interactions with water mole-
cules of solvation or with polymerase-based H-bond donors.
In contrast to H-bond acceptors introduced at the b posi-
tion,[6] this H-bond acceptor does not stabilize mispairs in
duplex DNA. While the ortho nitrogen atom does appear to
somewhat facilitate dGTP mispair synthesis, the mispair is
synthesized only inefficiently and, in fact, not significantly
faster than mispairs among the natural nucleotides.[12] More
problematic is the insertion of dATP opposite 2Py, which is
probably mediated by hydrophobic interactions. However,
dATP insertion opposite 2Py is less efficient than with other
scaffolds that have already been successfully optimized by
derivatization.[7,3b,c] Combination of these modifications with
ortho aza substitution may result in the simultaneous
optimization of interbase interactions and nucleobase–poly-
merase interactions and it should result in unnatural base
pairs that are both more stable and efficiently replicated.
Table 3: Triphosphate insertion opposite pyridyl analogues in the
template.[a]
5’-dTAATACGACTCACTATAGGGAGA
3’-dATTATGCTGAGTGATATCCCTCTXGCTAGGTTACGGCAGGATCGC
fldYTP
5’-dTAATACGACTCACTATAGGGAGAY
3’-dATTATGCTGAGTGATATCCCTCTXGCTAGGTTACGGCAGGATCGC
X
Y
kcat [minÀ1
]
KM [mm]
kcat/KM [mÀ1 minÀ1
]
2Py
2Py
A
C
G
T
A
C
G
T
A
C
0.92Æ0.16
3.6Æ1.2
0.16Æ0.02
0.8Æ0.3
0.16Æ0.06
6.8Æ0.9
n.d.[b]
149Æ12
18Æ2
98Æ30
41Æ9
39Æ9
25Æ0.5
n.d.[b]
6.2103
2.0105
3.2103
2.0104
4.1103
2.7105
<1.0103
1.9105
6.5103
5.5105
<1.0103
<1.0103
3.3104
3Py
4Py
11Æ3
58Æ13
83Æ15
20Æ6
n.d.[b]
0.54Æ0.05
11Æ3
n.d.[b]
G
T
n.d.[b]
n.d.[b]
1.6Æ0.3
49Æ16
[a] See the Supporting Information for experimental details. [b] Rates
were too slow for the determination of kcat and KM independently.
Received: June 28, 2006
The self pair is synthesized with a second-order rate constant
of 6.2 103 mÀ1 minÀ1. While this synthesis is less efficient than
that of self pairs comprised of nucleobase analogues with a
large, extended aromatic surface area[3a] or more highly
substituted phenyl rings,[3b,c] it is actually high compared to
other minimally substituted scaffolds. For example, the BEN
and PYR self pairs are both synthesized at rates less than
103 mÀ1 minÀ1.[3c,6]
Keywords: DNA replication · hydrophobic effects · nucleobases ·
.
polymerases · pyridines
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We also examined the rates at which each natural
triphosphate is inserted opposite each pyridyl analogue
(Table 3). As with most small hydrophobic analogues, dATP
was the most efficiently incorporated deoxynucleoside tri-
phosphate (dNTP)opposite the pyridyl analogues. The rate of
dATP insertion showed little dependence on aza substitution
and ranged from only 2 105–5.5 105 mÀ1 minÀ1. The magni-
tude and template independence of the rate of dATP
insertion suggests that it results from nonspecific hydrophobic
interactions. dGTP was the next most efficiently inserted
natural triphosphate opposite 2Py and 3Py, but its insertion
was not detectable opposite 4Py. The rates are at least roughly
correlated with the expected ability of the pyridine moiety to
H-bond with guanosine (Scheme 2). The absolute rate at
which the 2Py:dG, PYR:dG (1.2 104 mÀ1 minÀ1), and
BEN:dG (1.6 103 mÀ1 minÀ1)mispairs are formed suggests
that while the ortho nitrogen atom does not form stable H-
bonds with dG in duplex DNA, it facilitates dGTP insertion,
although less efficiently than the meta nitrogen substituent of
3Py. dTTP misincorporation opposite 2Py and 3Py proceeded
with rates of ꢀ 5 103 mÀ1 minÀ1; however, its insertion was an
order of magnitude more efficient opposite 4Py. Perhaps the
same factors that stabilize the 4Py:dT mispair also contribute
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