Expanding the scope of replicable unnatural DNA: Stepwise optimization of a predominantly hydrophobic base pair

Article abstract of DOI:10.1021/ja312148q

As part of an ongoing effort to expand the genetic alphabet for in vitro and eventually in vivo applications, we have synthesized a wide variety of predominantly hydrophobic unnatural base pairs exemplified by d5SICS-dMMO2 and d5SICS-dNaM. When incorporated into DNA, the latter is replicated and transcribed with greater efficiency and fidelity than the former; however, previous optimization efforts identified the para and methoxy-distal meta positions of dMMO2 as particularly promising for further optimization. Here, we report the stepwise optimization of dMMO2 via the synthesis and evaluation of 18 novel para-derivatized analogs of dMMO2, followed by further derivatization and evaluation of the most promising analogs with meta substituents. Subject to size constraints, we find that para substituents can optimize replication via both steric and electronic effects and that meta methoxy groups are unfavorable, while fluoro substituents can be beneficial or deleterious depending on the para substituent. In addition, we find that improvements in the efficiency of unnatural triphosphate insertion translate most directly into higher fidelity replication. Importantly, we identify multiple, unique base pair derivatives that when incorporated into DNA are well replicated. The most promising, d5SICS-dFEMO, is replicated under some conditions with greater efficiency and fidelity than d5SICS-dNaM. These results clearly demonstrate the generality of hydrophobic forces for the control of base pairing within DNA, provide a wealth of new SAR data, and importantly identify multiple new candidates for eventual in vivo evaluation.

Full text of DOI:10.1021/ja312148q

Article
pubs.acs.org/JACS
Expanding the Scope of Replicable Unnatural DNA: Stepwise
Optimization of a Predominantly Hydrophobic Base Pair
Thomas Lavergne, Melissa Degardin, Denis A. Malyshev, Henry T. Quach, Kirandeep Dhami,
́
Phillip Ordoukhanian, and Floyd E. Romesberg*
Department of Chemistry and Center for Protein and Nucleic Acid Research, The Scripps Research Institute,
10550 North Torrey Pines Road, La Jolla, California 92037, United States
S
* Supporting Information
ABSTRACT: As part of an ongoing effort to expand the ge-
netic alphabet for in vitro and eventually in vivo applications,
we have synthesized a wide variety of predominantly hydro-
phobic unnatural base pairs exemplified by d5SICS-dMMO2
and d5SICS-dNaM. When incorporated into DNA, the latter is
replicated and transcribed with greater efficiency and fidelity
than the former; however, previous optimization efforts iden-
tified the para and methoxy-distal meta positions of dMMO2
as particularly promising for further optimization. Here, we
report the stepwise optimization of dMMO2 via the synthesis
and evaluation of 18 novel para-derivatized analogs of dMMO2, followed by further derivatization and evaluation of the most
promising analogs with meta substituents. Subject to size constraints, we find that para substituents can optimize replication via
both steric and electronic effects and that meta methoxy groups are unfavorable, while fluoro substituents can be beneficial or
deleterious depending on the para substituent. In addition, we find that improvements in the efficiency of unnatural triphosphate
insertion translate most directly into higher fidelity replication. Importantly, we identify multiple, unique base pair derivatives that
when incorporated into DNA are well replicated. The most promising, d5SICS-dFEMO, is replicated under some conditions
with greater efficiency and fidelity than d5SICS-dNaM. These results clearly demonstrate the generality of hydrophobic forces
for the control of base pairing within DNA, provide a wealth of new SAR data, and importantly identify multiple new candidates
for eventual in vivo evaluation.
1. INTRODUCTION
With the long-term goal of expanding the genetic code, we1−4
and others5−7 have worked toward the identification of unnatural
nucleotides that stably pair within duplex DNA as well as
during replication and transcription, and thus constitute an
unnatural base pair. We have identified a class of unnatural
base pairs, exemplified by d5SICS-dMMO2 and d5SICS-
dNaM (Figure 1A), that are both efficiently replicated2,8,9 and
efficiently transcribed.10 From a conceptual perspective, this
efficient replication and transcription is of particular interest
because these processes are mediated only by hydrophobic and
packing forces between nucleobases that have no structural
homology to their natural counterparts. Overall, d5SICS-dNaM
is replicated and transcribed more efficiently than d5SICS-
dMMO2, and is also the only unnatural base pair shown to be
Figure 1. (A) Unnatural base pairs d5SICS-dMMO2 and d5SICS-dNaM.
efficiently replicated in a sequence-independent manner during
(B) dMMO2 derivatives, dDMO, dNMO1, dPMO1, and d5FM. Only
PCR;2 however, the individual steps of replication are not
nucleobase analogs shown with sugar and phosphate omitted for clarity.
equally efficient. For example, incorporation of dMMO2TP
opposite d5SICS is less efficient than incorporation of
dNaMTP, but continued extension of a primer terminating
dNaM,8−11 the relative contributions of efficient unnatural
triphosphate incorporation and extension to the overall
with dNaM by incorporation of the next correct triphosphate is
slower than that of a primer terminating with dMMO2. While
past SAR studies have demonstrated that replication is most
limited by the synthesis of the strand containing dMMO2 or
Received: December 12, 2012
Published: April 2, 2013
© 2013 American Chemical Society
5408
dx.doi.org/10.1021/ja312148q | J. Am. Chem. Soc. 2013, 135, 5408−5419

Journal of the American Chemical Society
Article
Figure 2. (A) Eighteen mono para-substituted analogs of dMMO2. (B) Five meta-, para-disubstituted analogs of dMMO2. Only nucleobase analogs
shown with sugar and phosphate omitted for clarity.
efficiency and fidelity are not well understood. Thus, both
dMMO2 and dNaM remain promising partners for d5SICS,
but the simpler and more atom-economical scaffold of dMMO2
makes it a particularly promising scaffold for further
optimization.
A wealth of SAR data was generated and several well replicated
derivative base pairs were identified, including d5SICS-
dFEMO, which under some conditions is replicated better
than d5SICS-dNaM. These results further demonstrate the
robustness and generality of hydrophobic and packing forces
for the control of DNA replication and also further validate the
dMMO2 scaffold as a partner for d5SICS. Moreover, several of
the newly identified unnatural base pairs are not only well
replicated but also have varying physicochemical properties that
may eventually facilitate replication in vivo.
Previous structure−activity relationship (SAR) data indicate
that the ortho methoxy group of the dMMO2 scaffold is
necessary for efficient replication,8,12,13 and that substituents at
the adjacent meta position are not well tolerated.14−16 Thus,
modification at the para- and remaining meta-position of
the dMMO2 scaffold appears to be most promising for
optimization. Previous SAR studies also suggest that
modifications at the para position generally have larger effects,
for example, dDMOTP, dNMO1TP, and dPMO1TP (Figure 1B)
are inserted opposite d5SICS more efficiently than dMMO2TP,9,17
but those at the meta position can also be beneficial, for
example, after incorporation of the corresponding triphos-
phate, d5FM (Figure 1B) is more efficiently extended than
dMMO2.10 Nonetheless, all of the resulting unnatural pairs are
still replicated significantly less efficiently than d5SICS-dNaM.
Nowhere has the optimization of synthetic molecules for
biological function been more successful than in medicinal
chemistry, which traditionally relies on the synthesis of
derivatives in conjunction with efficient assays for the rapid
identification of the most promising compounds and the
elucidation of SAR data for additional optimization efforts. To
emulate this approach, herein we report an optimized set of
divergent synthetic strategies to access derivatives of
dMMO2TP, as well as their efficient analysis via pre-steady-
state kinetics and PCR assays. We synthesized a small library of
novel paraderivatized dMMO2 analogs that, when combined
with dDMO, dNMO1, and dPMO1, provide a much more
complete survey of the potential of this site for optimization.
Several of the most promising analogs were then further
derivatized with meta fluorine or methoxy substituents, whose
characterization along with d5FM provides an initial analysis
of the effects of simultaneous meta- and para-derivatization.
2. RESULTS
2.1. Design and Synthesis of para-Substituted Deriva-
tives of dMMO2. We first designed 18 para-derivatized
dMMO2 analogs (Figure 2A) which, when combined with the
previously reported analogs, dDMO, dNMO1, and dPMO1,
provide a rather complete survey of steric and electronic effects.
Along with dPMO1, the bis-aromatic analogs dPhMO, dPyMO1,
dPyMO2, dTpMO1, dTpMO2, dFuMO1, dFuMO2, dPMO2,
and dPMO3 were designed to explore the effects of annular
substituents and the dIMO and dClMO derivatives were designed
to alter nucleobase bulk and electronics. The remainder of the
analogs, dPrMO, dEMO, dVMO, dCNMO, dZMO, dQMO
and dTfMO, were designed to help deconvolute the con-
tributions of sterics and electrostatics.
The unnatural nucleotides analogs were synthesized as
shown in Schemes 1−5. dQMO, dIMO and dClMO triphos-
phates were obtained from the previously reported precursor 19
(Scheme 1).18 Briefly, hydroxyl group protection followed
by hydrogenation afforded compound 2, which was then
sulfonated,19 coupled to acrolein via conjugate addition,
acidified to form the quinoline ring, and finally deprotected
with sodium methoxide to provide dQMO (3) in good yield.
Toward dIMO (4) and dClMO (5), 2 was subjected to
Sandmeyer iodination and chlorination, respectively, and then
deprotected. Free nucleosides 3−5 were converted to the
corresponding triphosphates 6−8 under Ludwig conditions,20
5409
dx.doi.org/10.1021/ja312148q | J. Am. Chem. Soc. 2013, 135, 5408−5419

Journal of the American Chemical Society
Article
a
Scheme 1
a
Conditions: (a) Toluyl chloride, pyridine, rt, 15 h, 59%; (b) 10% Pd/C, H2, EtOAc, NEt3, rt, 1 h, 91%; (c) (1) TsCl, pyr, CH2Cl2, rt, 40 min;
(2) acrolein, NEt3, MeOH, 0 °C → rt, 20 min, 85%; (d) HCl 3N, THF, 80 °C, 40 min, 80%; (e) HCl aq 6 M, NaNO2, KI, THF, 0 °C → rt, 2 h, 55%;
(f) HCl aq 6 M, NaNO2, CuCl, THF, 0 °C → 40 °C, 5 h, 23%; (g) MeONa 30% in MeOH, MeOH:CH2Cl2 8:2, 5 °C → rt, 30 min to1 h: 3, 90%; 4,
86%; 5, 96%; (h) tBuOK, THF, 70 °C, 3 h, 78%; (i) proton sponge, POCl3, PO(OMe)3, −15 °C → −10 °C, 3 h then Bu3N, (Bu3NH)2H2P2O7 in
DMF, −10 °C → 0 °C, 30 min then TEAB buffer (0.5M), rt, 10 min: 6, 27%; 7, 48%; 8, 57%.
a
Scheme 2
a
Conditions: (a) CuI, 70 °C, 16 h, 1,10-phenanthroline, KCF3B(OMe)3, DMSO, 70%; (b) Pd2dba3, CuI, AsPh3, vinyltributyltin, dioxane, 50 °C, 2 h,
72%; (c) K4Fe(CN)6, Pd(OAc)2, KF, TBAB, H2O, microwave-150 °C, 15 min, 55%; (d) NaN3, CuI, N,N′-dimethylethylenediamine, 90 °C, 45 min,
74%; (e) MeONa 30% in MeOH, MeOH/CH2Cl2 8/2, 5 °C → rt, 45 min to 1 h: 10, 85%; 11, 90%; 12, 79%; 13, 98%; (f) proton sponge, POCl3,
PO(OMe)3, −15 °C → −10 °C, 3 h then Bu3N, (Bu3NH)2H2P2O7 in DMF, −10 °C → 0 °C, 30 min then TEAB buffer (0.5 M), rt, 10 min: 14,
20%; 15, 36%; 16, 42%; 17, 25%.
and purified by anion exchange chromatography followed by
HPLC. The purity of each triphosphate was confirmed by 31P
NMR, HPLC, and MALDI-TOF MS (Supporting Information).
Nucleotides dTfMO, dVMO, dCNMO and dZMO were
obtained from the toluyl protected intermediate 9 as shown in
Scheme 2. Potassium (trifluoromethyl)trimethoxyborate was
used as a source of CF3 nucleophiles for the copper-catalyzed
trifluoromethylation,21 and deprotection yielded dTfMO (10).
Toward dVMO, we found that Suzuki-Miyaura cross-coupling
with vinyltrifluoroborate,22 palladium cross-coupling with
5410
dx.doi.org/10.1021/ja312148q | J. Am. Chem. Soc. 2013, 135, 5408−5419

Journal of the American Chemical Society
Article
a
Scheme 3
a
Conditions: (a) Pd(OAc)2, TPPTS, Cs2CO3, boronic acid derivative, H2O:ACN 2:1, 70 °C, 30 min, >70%; (b) Pd(OAc)2, TPPTS, CuI, NEt3,
H2O/ACN 2/1, 30 min, 27 propyne 70 °C, 70%, 28 triethylsilylacetylene 55 °C 65%; (c) NH4OH 30%, rt, 1 h, 28, 60% 2 steps.
vinylaluminium reagent23 or vinyltriethoxysilane,24 or Stille
cross-coupling with vinyltributyltin25 all resulted in the con-
version of the aromatic iodide (9) to its vinyl analog with good
yields. Because the Stille cross-coupling generated cleaner crude
material, we proceeded with this route, and the dVMO (11)
nucleoside was obtained after deprotection. Palladium-catalyzed
cyanation of the aryl iodide (9) using potassium hexacyano-
ferrate (II) in water and under microwave irradiation, followed
by deprotection yielded dCNMO (12).26 It is noteworthy that,
with this particular substrate, palladium-catalyzed cyanation in
organic solvent using zinc cyanide failed to give any desired
product and only low yields were obtained with copper cyanide.
Toward dZMO, the aromatic iodide of 9 was subjected to a
mild CuI/diamine catalyzed Ulmann type coupling with aqueous
sodium azide. The reaction proceeded cleanly to completion and
deprotection then provided dZMO (13) in good yield. Free
nucleosides 10−13 were converted to the corresponding
triphosphates 14−17 and purified as described above.
kinetics data less helpful for the optimization of processive
synthesis. Thus, we developed a higher throughput pre-steady-
state assay that is based on determining under a fixed set of
conditions the amount of a dMMO2TP analog and dCTP that
are added to a 23mer primer opposite their cognate nucleotides
in a 45mer template (containing d5SICS at position 24 and dG
at position 25) by the Klenow fragment of Escherichia coli DNA
polymerase I (Kf). The percent incorporation (%incorpora-
tion) of the unnatural triphosphate was defined as the ratio,
[24mer + 25mer]/[23mer + 24mer + 25mer], and the percent
extension (%extension) was defined as the ratio, [25mer]/
[24mer + 25mer], determined in the presence of saturating
concentrations of unnatural triphosphate.
We first explored DNA synthesis with relatively high
concentrations of unnatural triphosphate and dCTP (20 μM
each; Figure 3 and Table S1) and with reaction times of 10 s.
The triphosphates of dPhMO, dPyMO1, dPyMO2, dTpMO1,
dTpMO2, dFuMO1, dFuMO2, dPMO2, dPMO3, dPrMO, and
dEMO were readily obtained from the unprotected triphosphate 7
using aqueous Sonogashira or Suzuki-Miyaura cross-coupling
(Scheme 3). dPhMO to dPMO3 (18−26) were obtained using
a previously reported approach involving aqueous palladium
cross-coupling in the presence of a water-soluble sulfonated
triphenylphosphine ligand (TPPTS) and cesium carbonate with
quantitative conversion of the aromatic amine.27−32 Reaction
time and temperature were optimized to avoid triphosphate
degradation. dPrMO triphosphate (27) was obtained using
aqueous copper catalyzed Sonogashira coupling in presence of
TPPTS, triethylamine and a large excess of propyne gas. The
dEMO triphosphate (28) was obtained similarly by coupling
triethylsilylacetylene and freeing the alkyne with ammonia.
Each triphosphate was purified as described above.
2.2. Initial Pre-Steady-State Kinetic Analysis of para-
Modified Derivatives. In previous work, we employed
steady-state kinetics to analyze the various steps that contribute
to the replication of DNA containing an unnatural base pair,
including the rate at which the unnatural base pair is syn-
thesized (by incorporation of an unnatural triphosphate
opposite its cognate base in a template), and the rate at
which the nascent primer terminus is extended by incorpo-
ration of the next correct natural triphosphate. While such
experiments are time intensive, they provided critical
information about the synthesis of the unnatural base pairs,
which for the early and less efficiently replicated analogs was
required for optimization. In contrast, replication of the current
candidates is very efficient and under steady-state conditions
limited by product dissociation,33 rendering the steady-state
Figure 3. Values of %incorporation and %extension with 10 s reaction
times and 20 μM dMMO2 analog/20 μM dCTP.
Under these conditions, all of the reactions, including those
with dMMO2TP and dNaMTP, showed similar accumula-
tion of 24mer, confirming that incorporation is fast relative to
extension and that 20 μM of the unnatural triphosphate is
sufficient for saturation (further confirmed with reactions run
with 50 μM unnatural triphosphate, data not shown). In con-
trast, very different %extension values were observed in each
reaction. With dMMO2TP or dNaMTP at the primer terminus,
the %extension is 85%. Nine derivatives paired opposite d5SICS
5411
dx.doi.org/10.1021/ja312148q | J. Am. Chem. Soc. 2013, 135, 5408−5419

Journal of the American Chemical Society
Article
are extended significantly less efficiently, including dPhMO,
dTpMO1, dPyMO1, dTpMO2, dPMO1, dPyMO2, dPMO2,
dFuMO2, and dFuMO1. The four derivatives dNMO1, dPMO3,
dQMO, and dTfMO are extended more efficiently, but still
significantly less efficiently than dMMO2TP or dNaMTP.
Interestingly, the %extension of eight derivatives, including
dVMO, dIMO, dClMO, dCNMO, dZMO, dDMO, dPrMO,
and dEMO, is slightly greater than that of either dMMO2TP or
dNaMTP.
To further differentiate the unnatural triphosphates, we ex-
amined DNA synthesis in the presence of lower concentrations
of triphosphates (for incorporation, 1 μM for both unnatural
triphosphates and dCTP, and for extension, 20 μM unnatural
triphosphate and 1 μM dCTP; Figure 4 and Table S2. Under
significantly to moderately lower than dNaM, including,
dPhMO, dPyMO1, dTpMO2, dPyMO2, dTpMO1, dFuMO1,
dFuMO2, dPMO2, dPMO1, dNMO1, dPMO3, dQMO,
dTfMO, and dIMO, and three similar to dNaM, including
dPrMO, dCNMO, and dVMO. Interestingly, dClMO, dZMO,
and dEMO paired opposite d5SICS are extended with
efficiencies similar to dMMO2, while dDMO is extended
more efficiently.
2.3. More Stringent Pre-Steady-State Kinetic Analysis
of the Most Promising para-Modified Derivatives. On
the basis of the preliminary analysis described above, the seven
para substituted derivatives, dPrMO, dEMO, dIMO, dClMO,
dCNMO, dZMO, and dDMO, were selected for further anal-
ysis under more stringent conditions. We first measured DNA
synthesis with shorter reaction times (5 s), and with unnatural
triphosphate and dCTP concentrations maintained at 1 μM to
characterize unnatural triphosphate incorporation and at 20 and
1 μM, respectively, to characterize extension (Figure 5 and
Figure 4. Values of %incorporation and %extension with 10 s reaction
times and with 1 μM dMMO2 analog/1 μM dCTP for the
incorporation reactions and 20 μM dMMO2 analog/1 μM dCTP
for the extension reactions. Error bars shown are standard deviations
determined from three independent experiments.
Figure 5. Values of %incorporation and %extension with 5 s reaction
times and 1 μM dMMO2 analog/1 μM dCTP for the incorporation
reactions and 20 μM dMMO2 analog/1 μM dCTP for the extension
reactions. Error bars shown are standard deviations determined from
three independent experiments.
these conditions, the %incorporation values for dMMO2TP
and dNaMTP are 27% and 69%, respectively. As expected, a
much broader range of incorporation efficiencies were observed
with the different analogs (12% to 65%) than at high triphos-
phate concentrations. Five of the analogs are incorporated less
efficiently than dMMO2TP, including dPMO2TP, dPMO3TP,
dPyMO1TP, dPhMOTP, and dVMOTP, and sixteen are
inserted better, including, dPyMO2TP, dFuMO1TP,
dPMO1TP, dNMO1TP, dTpMO1TP, dFuMO2TP,
dTpMO2TP, dDMOTP, dTfMOTP, dPrMOTP, dEMOTP,
dClMOTP, dZMOTP, dQMOTP, dCNMOTP, and dI-
MOTP. While dQMOTP incorporation is more efficient
than dMMO2TP incorporation, it is less efficient than dNaM
incorporation, demonstrating that the added nitrogen sub-
stituent is not beneficial. Most interestingly, under these con-
ditions the %incorporation values for dEMOTP, dClMOTP,
dZMOTP, dQMOTP, dCNMOTP, and dIMOTP approach
that for dNaMTP.
Table S3). Under these conditions, the %incorporation values
for dMMO2TP and dNaMTP are 17% and 64%, respectively,
and the %extension values for the corresponding unnatural
primer termini are 30% and 23%, respectively. For each of the
derivative triphosphates, the %incorporation is greater than that
for dMMO2TP, with dIMOTP exhibiting the highest value of
52%. Three derivatives are extended less efficiently than dNaM,
including dIMOTP, dPrMO, and dCNMO; dZMO is extended
with an efficiency between dNaM and dMMO2; and dClMO,
dEMO, and dDMO are actually extended more efficiently than
either dMMO2 or dNaM.
We next examined synthesis with further reduced concen-
trations of triphosphates (0.2 μM unnatural triphosphate and
0.5 μM dCTP for incorporation, and 20 μM unnatural tripho-
sphate and 0.5 μM dCTP for extension) (Figure 6 and Table S4).
For reference, we note that, even under these challenging con-
ditions, the %incorporation and %extension of a dC-dG base
pair remain above 90%. Under these incorporation conditions,
At the reduced dCTP concentration, the %extension values
for dMMO2 or dNaM paired opposite d5SICS are 50% and
33%, respectively. Again, a wide variety of extension efficiencies
were observed for the different derivatives (Figure 4), with 14
5412
dx.doi.org/10.1021/ja312148q | J. Am. Chem. Soc. 2013, 135, 5408−5419

Journal of the American Chemical Society
Article
derivatives, dZMO, dEMO, and most notably dDMO, are
extended more efficiently than dMMO2.
2.4. Design, Synthesis, and Analysis of Five meta,
para-Disubstituted Derivatives. On the basis of the above-
described data and the potential for generating illuminating
SAR data, five para substituted derivatives were selected for
further derivatization with a meta fluoro or methoxy substituent,
generating dFIMOTP, dMIMOTP, dFEMOTP, dMEMOTP,
and dFDMOTP (Figure 2B). Because of its analogous substitu-
tion pattern, we also included the previously reported d5FMTP
derivative in the current analysis (Figure 1B).
dFIMO, dFDMO, and dFEMO were synthesized as shown
in Scheme 4. First, commercially available 2-fluoro-5-methox-
yaniline was protected and iodinated in the presence of a silver
salt in a nonprotic solvent to afford the anisidine 29. The
modified nucleoside 31 was then obtained in three steps via
Heck coupling of 29 and the 2′-deoxyribose glycal 30, followed
by sugar deprotection and selective reduction of the resulting 3′
keto group. Hydroxyl groups were protected with toluyl groups
and the Cbz group was removed by hydrogenation. dFIMO
(33) was prepared from 31 via a Sandmeyer iodination fol-
lowed by sugar deprotection. We note that due to the inherent
instability of the aryl diazonium intermediate, efficient
iodination required the simultaneous addition of sodium nitrite
and iodine salts. Analog dFDMO (34) was obtained from 31
via a copper-catalyzed coupling in neat methanol in the pres-
ence of 1,10-phenanthroline and cesium carbonate.34 Efficient
product formation required 6 h at 110 °C and microwave
irradiation, and even under these optimized conditions, a small
amount of the reduced 3-fluoroanisole nucleoside byproduct
was consistently detected. During the course of the reaction,
the toluyl groups were removed, and dFDMO (34) was ob-
tained after silica gel purification. Free nucleosides 33−34 were
converted to the corresponding triphosphates 35−36 and
purified as described above. The dFEMO triphosphate (37)
was obtained from the dFIMO triphosphate (35) using
Figure 6. Values of %incorporation and %extension with 10 s reaction
times and 0.2 μM dMMO2 analog/0.5 μM dCTP for incorporation
reactions and 20 μM dMMO2 analog/0.5 μM dCTP for extension
reactions. Error bars shown are standard deviations determined from
three independent experiments.
the %incorporation values for dMMO2TP and dNaMTP are
10% and 45%, respectively. Again, the %incorporation for each
derivative triphosphate is intermediate between those of
dMMO2TP and dNaMTP, with dIMOTP being the greatest.
Under these extension conditions, the pairs formed between
d5SICS and dNaMTP or dMMO2TP are extended with
%extensions of 22% and 35%, respectively. Two derivatives,
dIMO and dPrMO, are extended less efficiently than dNaM,
while dCNMO and dClMO are inserted with efficiencies inter-
mediate between those of dNaM and dMMO2, and lastly three
a
Scheme 4
a
Conditions: (a) CBz-Cl, NaHCO3, THF, rt, 1 h, 84%; (b) I2, Ag2SO4, ACN, - 20 °C, 40 min, 96%; (c) Pd(OAc)2, AsPh3, nBu3N, DMF, 70 °C,
15 h; (d) TBAF 1 M in THF, 0 °C → rt, 4 h, 54% 2 steps; (e) NaBH(OAc)3, AcOH, CH3CN, −4 °C, 1 h, 91%; (f) toluyl chloride, pyridine, rt, 3 h,
88%; (g) 10% Pd/C, H2, EtOAc, NEt3, rt, 1 h, 70%; (h) NaNO2, KI, HCl aq 6 M, THF, 0 °C → rt, 2 h, 40%; (i) MeONa 30% in MeOH,
MeOH:CH2Cl2 8:2, rt, 15 min, 92%; (j) CuI, 1,10-phenanthroline, Cs2CO3, MeOH, microwave-110 °C, 6 h, 46%; (k) proton sponge, POCl3,
PO(OMe)3, −15 °C → −10 °C, 3 h then Bu3N, (Bu3NH)2H2P2O7 in DMF, −10 °C → 0 °C, 30 min then TEAB buffer (0.5M), rt, 10 min: 35, 16%;
36, 20%; (l) Pd(OAc)2, 3,3′,3″-phosphinidynetris(benzenesulfonic acid) trisodium salt (TPPTS), CuI, triethylsilylacetylene, Et3N, H2O:ACN 2:1,
65°C, 30 min; (m) NH4OH 30%, rt, 1 h, 50% 2 steps.
5413
dx.doi.org/10.1021/ja312148q | J. Am. Chem. Soc. 2013, 135, 5408−5419

Journal of the American Chemical Society
Article
a
Scheme 5
a
Conditions: (a) I2, H5IO6, MeOH, 70 °C, 5 h, 79%; (b) Pd(OAc)2, AsPh3, nBu3N, DMF, 70 °C, 15 h; (c) TBAF 1 M in THF, 0 °C → rt, 1 h, 27%
2 steps; (d) NaBH(OAc)3, AcOH, CH3CN, −4 °C, 45 min, 88%; (e) proton sponge, POCl3, PO(OMe)3, −15 °C → −10 °C, 3 h then Bu3N,
(Bu3NH)2H2P2O7 in DMF, −10 °C → 0 °C, 30 min then TEAB buffer (0.5M), rt, 10 min, 27%; (f) Pd(OAc)2, TPPTS, CuI, triethylsilylacetylene,
Et3N, H2O:ACN 2:1, 65 °C, 30 min; (g) NH4OH 30%, rt, 1 h, 40% 2 steps.
aqueous copper catalyzed Sonogashira coupling in the presence
of triethylsilylacetylene, followed by removal of the triethylsilyl
protecting group as described above.
Table 1. PCR Amplification and Fidelity with OneTaq DNA
Polymerase
a
b
dMMO2 analog
amplification
fidelity
The dMIMO and dMEMO analogs were synthesized from
the commercially available 2,4-dimethoxybenzene via diiodina-
tion, as previously reported35 (Scheme 5). The modified
nucleoside 38 was then obtained in three steps via Heck
coupling with the 2′-deoxyribose glycal 30, followed by sugar
deprotection and selective reduction. Free nucleoside 38 was
then converted to the corresponding triphosphate 39 as
described above. The dMEMO triphosphate (40) was obtained
from 39 via an aqueous copper catalyzed Sonogashira coupling
in presence of triethylsilylacetylene followed by triethylsilyl
deprotection.
The incorporation and extension of the resulting six meta,
para-disubstituted derivatives were examined under each of the
pre-steady-state assay conditions described above (Figures 3−6).
We found that methoxy substitution in both cases examined
(dMIMO and dMEMO) significantly decreases both the
%incorporation and %extension, while the effects of fluoro
substitution are more variable. In the case of dFDMOTP, the
fluoro substituent dramatically reduces both %incorporation
and %extension (relative to dDMOTP). With dFIMOTP,
we found that the fluoro substituent increases incorporation
efficiency, but has little effect on extension (relative to
dIMOTP), while with d5FMTP, it has little effect on incorpora-
tion but significantly increases extension (relative to dMMO2TP).
Finally, with dFEMOTP, the fluoro substituent significantly
increases the efficiency of both incorporation and extension.
Importantly, under these pre-steady-state conditions, including
both unnatural triphosphate incorporation and extension,
d5SICS-dFEMO is more efficiently replicated than d5SICS-
dNaM.
2.5. PCR Analysis. To more fully evaluate replication, DNA
containing a dMMO2 analog paired opposite d5SICS was
amplified by PCR. Efficiency was characterized by monitoring
amplification level and fidelity (defined as unnatural base pair
retention per doubling) was determined by amplicon se-
quencing (Figures S62−S65). Initial assays were performed
with 100 pg of a previously reported DNA template (previously
referred to as D6,2,11 where the unnatural base pair is flanked
on each side by three randomized natural nucleotides, Supporting
Information), 100 μM unnatural triphosphate, and 200 μM of
each natural dNTP, a 60 s extension time, and OneTaq poly-
merase, which is a commercially available mixture of two family A
polymerases, exonuclease-negative Taq polymerase and exonu-
clease-positive DeepVent (Table 1). To facilitate this initial screen,
the DNA was subjected to only 14 cycles of amplification,
c
dPhMO
dPyMO1
dPyMO2
dTpMO1
dTpMO2
dFuMO1
dFuMO2
dPMO2
dPrMO
dEMO
3.1 × 102
2.6 × 102
2.2 × 102
0.4 × 102
0.8 × 102
3.3 × 102
1.8 × 102
3.0 × 102
6.0 × 102
7.1 × 102
5.3 × 102
5.0 × 102
6.0 × 102
6.3 × 102
6.8 × 102
4.6 × 102
5.6 × 102
5.4 × 102
5.0 × 102
6.3 × 102
3.2 × 102
7.5 × 102
3.7 × 102
4.7 × 102
5.4 × 102
6.2 × 102
7.9 × 102
6.4 × 102
14 × 102
<90
c
c
c
c
c
c
c
<90
<90
<90
<90
<90
<90
<90
97.0 0.3
98.48 0.04
97.41 0.17
91.57 0.12
99.23 0.05
98.9 0.3
96.89 0.08
97.2 0.2
98.2 0.2
98.99 0.07
95.7 0.3
98.7 0.2
94.3 0.4
98.6 0.4
95.0 0.8
97.6 0.3
99.85 0.13
97.49 0.01
96.6 0.3
96.3 0.5
dNMO1
dPMO1
dIMO
dClMO
dCNMO
dTfMO
dVMO
dZMO
dQMO
dFIMO
dMIMO
dFEMO
dMEMO
dFDMO
dNaM
dMMO2
dDMO
d5FM
d
e
-
n.d.
a
See Materials and Methods for experimental details. Error was
b
determined from three independent experiments. Fidelity (f) was
determined by sequencing (see Materials and Methods) and is defined
as the retention of the unnatural base pair per doubling, calculated as
R = fn, where R is the retention of the unnatural base pair, n is the
number of doublings, calculated as log2(A), and A is the amplification
level. Errors for f were propagated from those determined for R.
Unnatural base pair retention was below 50% and the fidelity was thus
estimated to be below 90%. Natural template was amplified without
unnatural base pair under identical conditions as a control. n.d: not
c
d
e
determined.
obviating the need for dilutions during the amplification
process. Under these conditions, DNA containing dMMO2-
d5SICS or d5SICS-dNaM is amplified ∼600-fold (which is 2.5-
fold lower than the analogous DNA containing a natural dA-dT
5414
dx.doi.org/10.1021/ja312148q | J. Am. Chem. Soc. 2013, 135, 5408−5419

Journal of the American Chemical Society
Article
base pair at the same position) and with fidelities of 97.5% and
99.9%, respectively. DNA containing d5SICS paired opposite
one of the 10 derivatives dPhMO-dPMO3 is amplified with
only modest efficiency and fidelity. DNA containing d5SICS
paired opposite any of the remaining derivatives, except
dMIMO, dMEMO, and dFDMO, is amplified between 500-
and 800-fold, but with variable fidelity. The fidelity with DNA
containing dPMO1 is very low, while that with dMIMO,
dMEMO, dQMO, d5FM, dDMO, dCNMO, or dPrMO is
better, but still less than that with dMMO2. DNA containing
dTfMO or dNMO1, or dFDMO is amplified with similar
fidelity as that containing dMMO2, while DNA with dVMO,
dEMO, dFEMO, dFIMO, dClMO, or dZMO is amplified with
higher fidelity than that containing dMMO2. Under these
conditions, DNA containing d5SICS-dIMO is amplified with a
fidelity approaching that of DNA containing d5SICS-dNaM.
Previously, we reported that an optimal balance between
polymerization and 3′-5′ exonuclease activity is important for
the high fidelity amplification of DNA containing d5SICS-
dNaM.2 To determine if proofreading similarly contributes to
the replication of the derivatives explored here, we repeated the
amplifications for a subset of the analogs with Taq polymerase
alone, under conditions expected to emphasize differences that
included both higher amplification (starting with 10 pg of
template), and shorter extension times (15 s) (Table 2). Under
taining dCNMO, dIMO, dClMO, dZMO, or dEMO is better
amplified, but still not amplified as well as DNA containing
dNaM. However, under these conditions, DNA containing
dFEMO or dFIMO is amplified with fidelities approaching that
of DNA containing dNaM.
With the data supporting the importance of exonuclease
activity, we returned to OneTaq-mediated amplification and
examined the 1013-fold amplification of a subset of the analogs
(Table 3). Under these conditions, DNA containing dNMO1
Table 3. PCR Amplification and Fidelity with OneTaq DNA
a
Polymerase and High Amplification
b
dMMO2 analog
amplification
fidelity (sequencing)
dEMO
dNMO1
dIMO
1.4 × 1013
1.5 × 1013
1.3 × 1013
1.5 × 1013
1.5 × 1013
1.5 × 1013
1.5 × 1013
1.1 × 1013
1.5 × 1013
0.9 × 1013
2.7 × 1013
98.55 0.16
c
<96
98.3 0.4
98.2 0.3
97.4 0.3
dClMO
dCNMO
dVMO
dZMO
dFIMO
dFEMO
dNaM
c
<96
98.4 0.3
98.74 0.05
98.77 0.08
99.92 0.02
d
e
-
n.d.
a
See Materials and Methods for experimental details. Error was
b
Table 2. PCR Amplification and Fidelity with Taq DNA
determined from three independent experiments. Fidelity (f) was
determined by sequencing (see Materials and Methods) and is defined
as the retention of the unnatural base pair per doubling, calculated as
R = fn, where R is the retention of the unnatural base pair, n is the
number of doublings, calculated as log2(A), and A is the amplification
level. Errors for f were propagated from those determined for R.
a
Polymerase
b
dMMO2 analog
amplification
fidelity (sequencing)
c
dPrMO
dEMO
dNMO1
dIMO
3.7 × 103
6.0 × 103
3.4 × 103
4.2 × 103
5.9 × 103
5.0 × 103
3.0 × 103
2.9 × 103
4.2 × 103
2.9 × 103
4.4 × 103
6.9 × 103
2.2 × 103
3.7 × 103
2.9 × 103
1.1 × 103
3.2 × 103
29 × 103
<85
93.4 1.4
c
c
Unnatural base pair retention was below 50% and the fidelity was thus
<85
d
estimated to be below 96%. Natural template was amplified without
unnatural base pair under identical conditions as a control. n.d.: not
90.88 0.13
91.4 1.1
e
dClMO
dCNMO
dTfMO
dVMO
dZMO
dQMO
dFIMO
dFEMO
dFDMO
dNaM
determined.
88
4
c
c
<85
<85
or dVMO paired opposite d5SICS is not replicated well; DNA
containing dCNMO, dClMO, dIMO, dZMO, or dEMO, is
better replicated; and DNA containing d5SICS-dFIMO or
d5SICS-dFEMO is replicated with a fidelity approaching that
of d5SICS-dNaM.
91.69 0.12
c
<85
96.4 0.9
95.8 0.5
c
<85
In the OneTaq system, DNA is mainly replicated by Taq (a
family A polymerase36,37), while DeepVent (a family B poly-
merase36,37) is mainly responsible for proofreading. To explore
replication by a family B polymerase alone, PCR amplifications
were performed with KOD polymerase and a select set of the
analogs (Table 4). KOD clearly replicates d5SICS-dNaM with
lower fidelity than either OneTaq or Taq, and replicates the
pairs with dIMO and dFIMO with even lower fidelity. How-
ever, DNA containing dZMO, dClMO, dEMO, dCNMO, or
especially dFEMO paired opposite d5SICS is replicated better
than with dNaM paired opposite d5SICS. The d5SICS-dFEMO
pair is especially noteworthy, as unlike the other pairs, its replication
with the family B polymerase is virtually as efficient and high fidelity
as replication with the A family polymerases.
98.11 0.03
c
dMMO2
dDMO
d5FM
<85
c
<85
c
<85
d
e
-
n.d.
a
See Materials and Methods for experimental details. Error was
b
determined from three independent experiments. Fidelity (f) was
determined by sequencing (see Materials and Methods) and is defined
as the retention of the unnatural base pair per doubling, calculated as
R = fn, where R is the retention of the unnatural base pair, n is the
number of doublings, calculated as log2(A), and A is the amplification
level. Errors for f were propagated from those determined for R.
c
Unnatural base pair retention was below 50% and the fidelity was thus
d
estimated to be below 85%. Natural template was amplified without
unnatural base pair under identical conditions as a control. n.d.: not
e
3. DISCUSSION
determined.
Following the identification of d5SICS-dMMO2 from a screen
of 3600 candidate hydrophobic unnatural base pairs and an
initial round of optimization,8 we focused our optimization
efforts on the para position of dMMO2. These efforts even-
tually yielded d5SICS-dDMO17 and d5SICS-dNaM,9,10 with
these conditions, d5SICS-dNaM is amplified with reduced
but still reasonable fidelity. However, neither DNA containing
dMMO2 nor that containing dPrMO, dNMO1, dTfMO,
dVMO, dQMO, dDMO, or d5FM is well amplified. DNA con-
5415
dx.doi.org/10.1021/ja312148q | J. Am. Chem. Soc. 2013, 135, 5408−5419

Journal of the American Chemical Society
Article
mandates deintercalation for continued primer extension.3,10
Thus, this class of derivatives does not appear promising.
To explore the effects of increased aromatic surface area in
the absence of a bicyclic nucleobase scaffold, para propynyl,
ethynyl, and vinyl substituents were explored with dPrMO,
dEMO, and dVMO, respectively. In addition, the effects of
altered structure and electronics were explored with dZMO and
dCNMO. The vinyl substituent was deleterious for both the
incorporation and extension steps of replication. In contrast,
all of the remaining substituents significantly increased the
efficiency of incorporation, although the increase was less
pronounced at lower triphosphate concentrations. Thus, the
data suggest that increased aromatic surface area and/or hydro-
phobicity, possibly subject to certain steric constraints, favor
efficient incorporation, and that relative to dNaM, this results
from an increase in the affinity of the polymerase for the
triphosphate. Relative to dMMO2, the ethinyl and azide
substituents have little effect on extension, and the propynyl
and cyano groups reduce efficiency, but apparently not due to
effects on the binding of dCTP. These effects may result from a
combination of steric and electronic factors, both between the
pairing nucleobases and with the polymerase. Whatever the
origins of the observed effects, with the exception of the vinyl
group, these aliphatic and heteroatom-modified para sub-
stituents appear to be promising for the optimization of
unnatural triphosphate incorporation.
The strongly electron withdrawing para nitro substituent
of dNMO1TP had only a small effect on the efficiency of
triphosphate incorporation opposite d5SICS, but dramatically
reduced extension efficiency of the resulting base pair. In con-
trast, the weaker electron withdrawing para halogen sub-
stituents, especially the iodo substituent, significantly increased
incorporation efficiency. In fact, at all but the lowest triphos-
phate concentrations examined, dIMO is inserted opposite
d5SICS almost as efficiently as dNaM. However, relative to
dNaM, the effects were somewhat attenuated at the lowest
triphosphate concentrations (0.2 μM), again suggesting that the
halogenated derivatives bind with an elevated KD. The chloro
substituent had little effect on extension, while the iodo
decreased it somewhat. As with the aliphatic and heteroatom-
derivatized analogs discussed above, halogens appear to be
promising para substituents for the optimization of triphos-
phate incorporation.
Table 4. PCR Amplification and Fidelity with KOD DNA
Polymerase
a
b
dMMO2 analog
amplification
fidelity (sequencing)
dEMO
dIMO
2.2 × 102
1.2 × 102
2.6 × 102
3.0 × 102
2.1 × 102
1.3 × 102
4.6 × 102
1.7 × 102
52 × 102
93.8 0.3
c
<85
dClMO
dCNMO
dZMO
dFIMO
dFEMO
dNaM
93.10 0.01
95.48 0.07
92.5 0.5
87.1 0.8
97.4 0.4
91.7 0.2
d
e
-
n.d.
a
See Materials and Methods for experimental details. Error was
b
determined from three independent experiments. Fidelity (f) was
determined by sequencing (see Materials and Methods) and is defined
as the retention of the unnatural base pair per doubling, calculated as
R = fn, where R is the retention of the unnatural base pair, n is the
number of doublings, calculated as log2(A), and A is the amplification
level. Errors for f were propagated from those determined for R.
c
Unnatural base pair retention was below 50% and the fidelity was thus
d
estimated to be below 85%. Natural template was amplified without
unnatural base pair under identical conditions as a control. n.d.: not
e
determined.
replication of the latter proceeding with the greatest efficiency
and highest fidelity, sufficiently so that it is functionally
equivalent to a natural base pair for PCR applications.2 How-
ever, optimization efforts also suggested that meta substituents
of the dMMO2 scaffold, such as fluorine, could optimize
replication.10,14 Nonetheless, it remained to be determined just
which substituents were optimal, whether substituents at both
positions would interact additively or synergistically, and whether
substituents might be identified that result in a dMMO2 derivative
that when paired with d5SICS is replicated as efficiently as
d5SICS-dNaM. To address these questions, we synthesized a
diverse set of para-derivatized dMMO2TP analogs that explore
a wide variety of structural and physicochemical variations, and
we developed pre-steady-state and PCR assays for their rapid
characterization. Following this initial optimization, several
derivatized nucleotides were selected based on their optimized
replication or their promise to provide illuminating SAR data
for a second phase of diversification via a meta methoxy or
fluoro substituent.
In both contexts examined, (dMIMO and dMEMO), a meta
methoxy substituent significantly decreased the efficiency of
both incorporation and extension. The effects were somewhat
smaller at low triphosphate concentrations, suggesting that
the methoxy substituents increase the affinity with which both
triphosphates bind. In addition, the effects were largely
independent of the para substituent. Because any mesomeric
effects should increase the electron density of the ortho
methoxy group, which at least for extension should be
favorable,8,12 the data suggest that the effects may result from
forced desolvation of the meta substituent. Regardless, the
meta-methoxy substituent is deleterious and will not be
included in future optimization efforts.
Very different effects were observed for a meta fluorine in the
four contexts examined (dFIMO, dFEMO, d5FM, and
dFDMO). In the case of dFDMO (relative to dDMO), the
efficiency of both incorporation and extension are reduced, at
least in part due to reduced natural and unnatural triphosphate
binding. For d5FM (relative to dMMO2), the efficiency of
extension is selectively increased, at least in part due to an
3.1. SAR Analysis. One of the goals of the present study
was to collect SAR data for both the incorporation of a
dMMO2TP analog opposite d5SICS, and the extension of the
resulting base pair. In previous efforts to optimize dMMO2, we
explored several bicyclic derivatives, such as dPMO1, which
as a triphosphate under steady-state conditions is inserted
opposite d5SICS slightly better than dMMO2TP.9 Large
differences in %incorporation were observed with the bicyclic
derivatives examined in the current study, with the best inserted
being the quinolone derivative, dQMOTP, followed by the
thiophene analogs dTpMO1TP and dTpMO2TP, and the
furan and pyrrole derivatives, dFuMO1TP, dFuMO2TP, and
dPyMO2TP. Clearly heteroatom substitution can have a
significant impact, for example, dPhMOTP and dPyMO1TP
are inserted much less efficiently than dPyMO2. While large
variations were observed in the rates of insertion of the bicyclic
derivatives opposite d5SICS, all of them effectively act as chain
terminators, due to very poor continued primer extension. This
likely results from increased interstrand intercalation between
the nucleobases, which may favor triphosphate insertion but
5416
dx.doi.org/10.1021/ja312148q | J. Am. Chem. Soc. 2013, 135, 5408−5419

Journal of the American Chemical Society
Article
increased affinity for natural triphosphate binding. For dFIMO
(relative to dIMO), the efficiency of incorporation and exten-
sion is marginally increased. Finally, for dFEMO (relative to
dEMO) the efficiency of both incorporation and extension
is increased significantly, at least in part due to increased
triphosphate binding. Thus, with an adjacent para methoxy
substituent, the meta fluorine substituent is deleterious, but
when adjacent to an iodo, methyl, or ethynyl substituent,
the meta fluorine substituent is neutral or beneficial. Clearly the
effects are not simply related to the size of the substituent.
The effects may be rooted in more subtle steric factors or in the
unique electron donating ability of the methoxy group. Subtle
and difficult to rationalize effects of nucleobase modifica-
tion have been observed with other analogs.38,39 Whatever the
detailed origins of the effects, the data clearly reveal that,
depending on the nature of the para substituent, a meta fluoro
substituent may be distinctly beneficial, especially for the
optimization of extension.
base pairs. While KOD is generally less optimal, with this B
family polymerase d5SICS-dFEMO is actually replicated better
than d5SICS-dNaM. This may result from the unique
mechanism for binding and delivering triphosphates to the
KOD active site that is based on electrostatic interactions
between the negatively charged triphosphate and basic residues
of the polymerase fingers domain.40 Moreover, KOD is highly
processive, suggesting that it might have an inherently high
affinity for DNA and/or triphosphates,41 possibly allowing
some perturbations to be tolerated. However, the other analogs
are not as well replicated as d5SICS-dFEMO, suggesting that
unique aspects of its structure or physiochemical properties are
especially compatible with KOD. Further exploration of the
relative replicability of d5SICS-dNaM and d5SICS-dFEMO
with different polymerases should not only illuminate the
differences in the potential substrate repertoires of different
polymerases, but should also help to define the determinants of
general replication and facilitate further optimization of the
unnatural base pair.
The data reveal that several of the para-derivatized dMMO2
derivatives form pairs with d5SICS that are PCR amplified with
reasonable efficiency and fidelity. While the effects of meta
methoxy substitution were not fully evaluated due to their poor
performance, it is clear that just as with the pre-steady-state
assays, the meta fluoro-substituents of dFIMO and dFEMO
improve amplification. When more fully comparing the kinetic
and PCR data, an absolute correlation is not expected as the
former reflects only one strand context of DNA synthesis.
Nonetheless, previous work suggests that the effects of
substituents in the context characterized (i.e., incorporation
of dMMO2TP analogs opposite d5SICS in the template) tend
to be larger than in the opposite context (i.e., with dMMO2
analogs in the template),8−11 and thus strong correlations
might persist. This is not the case with amplification efficiency.
All of the duplexes examined were amplified with an efficiency
within 2-fold of one another, and within ∼2−3-fold, 4−8-fold,
or 10−40-fold of that containing a natural base pair with
OneTaq, Taq, or KOD, respectively. This may result, at least in
part, from the relatively long extension times employed (1 min
for the OneTaq- and KOD-mediated amplifications). However,
there are more significant differences in fidelity. The exact
values of amplification fidelity in the cases where it is low are
not accurate (due to the experimental challenges of determin-
ing the level of unnatural base pair retention when it is very
low), and thus, we limited our analysis to only those analogs
that were generally replicated with higher fidelity and used the
data from the higher OneTaq amplification. Interestingly, a
clear correlation between %incorporation and fidelity is
observed, with correlation coefficients of 0.79, 0.82, 0.51, and
0.65, for the data from Tables 1−4, respectively (Figure S79).
Such a correlation is clearly expected in the limit of low or no
proofreading activity (3′-5′ exonuclease activity), which
suggests that exonucleolytic removal of an unnatural nucleotide
at a primer terminus may be inefficient. This conclusion is con-
sistent with the reduced fidelities observed during amplification
with Taq alone, and with our previous demonstration that
fidelity increased with increases in the ratio of polymerase
proofreading to extension activity.2 While this model requires
further investigation, the observed correlation suggests that
further efforts toward optimization of unnatural base pair
replication should focus on improving the rates of triphosphate
incorporation.
3.2. Progress toward Expansion of the Genetic Alphabet.
A primary goal of the present study was to determine if the
dMMO2 scaffold could be optimized as a partner for d5SICS.
Clearly, this goal was met by the identification of d5SICS-
dEMO, d5SICS-dFIMO, and d5SICS-dFEMO, which are
significantly better replicated than is d5SICS-dMMO2. In
addition, we note that the PCR experiments appear to suggest
that the replication of the analogs examined here is not strongly
sequence-dependent. This is based on an inspection of the
sequencing traces before and after amplification (the three
natural nucleotides flanking the unnatural base in the templates
employed pair were randomized). However, this data is
qualitative and the identification of any replication biases
imposed by the unnatural base pairs must await detailed char-
acterization. Future efforts will also focus on the character-
ization of mutation induced by insertion of an unnatural
triphosphate opposite a natural nucleotide. In addition, based
on the kinetic and PCR data, it appears that several mono
substituted para-derivatives not further explored by derivatiza-
tion here, including dZMO, dCNMO, and dClMO, merit
further exploration as scaffolds, as well. From a conceptual
perspective, especially when combined with other reported
hydrophobic unnatural base pairs that are well replicated,42,43
the optimizability and apparent robustness of the dMMO2
scaffold attests to the generality of hydrophobic and packing
interactions as forces that are capable of controlling the efficient
and high fidelity replication of DNA.
An immediate use for replicable unnatural base pairs is the
site-specific labeling of DNA within a PCR-amplifiable format
for in vitro applications ranging from basic biophysics to SELEX
and materials fabrication. The different dMMO2 analogs bear
a variety of functional groups that are interesting for such
applications. For example, F19 labeling of dFEMO provides an
NMR handle for characterization, the azido and cyano groups
of dZMO and dCNMO, respectively, provide IR probes with
unique absorptions,44,45 the iodo group of dIMO provides a
handle for bioconjugation via cross-coupling,46 and the azido
and alkyne substituents of dZMO, dEMO, and dFEMO
provide handles for bioconjugation via click chemistry.47,48
Efforts toward such applications are currently in progress.
A long-term goal of the effort to develop unnatural base pairs
is the expansion of the genetic alphabet in vivo and the crea-
tion of a semisynthetic organism with increased potential for
information storage and retrieval. However, in addition to
In agreement with previous results,2 OneTaq appears to be
optimal for the replication of DNA containing the unnatural
5417
dx.doi.org/10.1021/ja312148q | J. Am. Chem. Soc. 2013, 135, 5408−5419

Journal of the American Chemical Society
Article
efficient and high fidelity replication, the demands of the in vivo
environment include additional factors, such as substrate
uptake, localization within the cell, and off target protein
binding. These challenges are similar to those faced in drug
discovery, as drug candidates must possess, in addition to
suitable biochemical properties, favorable pharmacokinetic
properties. Such properties are scaffold-dependent but often
unpredictable, and thus, similar to efforts to develop any drug,
efforts to develop an unnatural base pair that is replicable in vivo
will be bolstered by the availability of multiple lead compounds
based on different scaffolds. The diversification of the dMMO2
scaffold into several new scaffolds that pair well with d5SICS is
in this regard of particular importance.
AUTHOR INFORMATION
■
Corresponding Author
floyd@scripps.edu
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by the National Institutes of Health
(GM 60005 to F.E.R).
REFERENCES
■
(1) McMinn, D. L.; Ogawa, A. K.; Wu, Y.; Liu, J.; Schultz, P. G.;
Romesberg, F. E. J. Am. Chem. Soc. 1999, 121, 11585−11586.
(2) Malyshev, D. A.; Dhami, K.; Quach, H. T.; Lavergne, T.;
Ordoukhanian, P.; Torkamani, A.; Romesberg, F. E. Proc. Natl. Acad.
Sci. U.S.A. 2012, 109, 12005−12010.
(3) Betz, K.; Malyshev, D. A.; Lavergne, T.; Welte, W.; Diederichs,
K.; Dwyer, T. J.; Ordoukhanian, P.; Romesberg, F. E.; Marx, A. Nat.
Chem. Biol. 2012, 8, 612−614.
(4) Seo, Y. J.; Malyshev, D. A.; Lavergne, T.; Ordoukhanian, P.;
Romesberg, F. E. J. Am. Chem. Soc. 2011, 133, 19878−19888.
(5) Yang, Z.; Chen, F.; Alvarado, J. B.; Benner, S. A. J. Am. Chem. Soc.
2011, 133, 15105−15112.
4. MATERIALS AND METHODS
4.1. General Synthetic Methods. Synthetic details and
compound characterization are provided in the Supporting Informa-
tion.
4.2. Gel-Based Incorporation/Extension Assay. Primer oligo-
nucleotides (Integrated DNA Technologies) were 5′-radiolabeled with
T4 polynucleotide kinase (New England Biolabs; Ipswich, MA) and
[γ-32P]-ATP (Perkin-Elmer) and annealed to template oligonucleo-
tides10 by heating to 95 °C followed by slow cooling to room
temperature. Reactions were initiated by adding a solution of
2× dNTP and dXTP solution (5 μL) to a solution containing polymerase
(73.53 nM) and primer:template (40 nM) in 5 μL Klenow reaction
buffer (50 mM Tris-HCl, pH 7.5, 10 mM DTT and 50 μg/mL
acetylated BSA). After incubation at 25 °C for 5−10 s, reactions were
quenched with 20 μL of loading dye (95% formamide, 20 mM EDTA,
and sufficient amounts of bromophenol blue and xylene cyanol).
Reaction products were resolved by 15% polyacrylamide gel
electrophoresis, and gel band intensities corresponding to the
extended and unextended primers were quantified by phosphorimag-
ing (Storm Imager, Molecular Dynamics) and Quantity One (Bio-
Rad) software. Except for the most permissive conditions, the reported
values are the average and standard deviation of three independent
determinations (see also Tables S1−S4).
4.3. PCR Assay. The synthesis of the DNA duplex used as a
template was described previously, where it was referred to as template
D6.11 The sequence of the d5SICS template strand is 5′-d-
GAAATTAATA CGACTCACTA TAGGGTTAAG CTTAACTTTA
AGAAGGAGAT TTACTATGGG TCCCGNNN5SICSN NNC-
GTCTGGT GAATTCCAAG TGCTAGCGCA TGTAATAACC
CGGGTCATAG CTGTTTCCTGTGTG-3′, where N is randomized
nucleotide and primer regions are underlined. OneTaq and Taq
enzymes were obtained from New England Biolabs and KOD Hot
Start DNA Polymerase was obtained from Novagen/EMD Millipore
Biosciences (Billerica, MA). PCR amplifications were performed in a
total volume of 25 μL and with conditions specific for each assay as
described in Table S5. After amplification, a 5 μL aliquot was analyzed
on a 2% agarose gel to confirm amplicon size (134 bp). The remaining
solution was purified by spin-column (DNA Clean and Concentrator-
5; Zymo Research, Irvine, CA), quantified by fluorescent dye binding
(Quant-iT dsDNA HS Assay kit, Invitrogen), and sequenced on a
3730 DNA Analyzer (Applied Biosystems). Fidelity was determined as
the average %retention of the unnatural base pair per doubling as
described in the Supporting Information.
(6) Kaul, C.; Muller, M.; Wagner, M.; Schneider, S.; Carell, T. Nat.
Chem. 2011, 3, 794−800.
(7) Kimoto, M.; Kawai, R.; Mitsui, T.; Yokoyama, S.; Hirao, I. Nucleic
Acids Res. 2009, 37, e14.
(8) Leconte, A. M.; Hwang, G. T.; Matsuda, S.; Capek, P.; Hari, Y.;
Romesberg, F. E. J. Am. Chem. Soc. 2008, 130, 2336−2343.
(9) Lavergne, T.; Malyshev, D. A.; Romesberg, F. E. Chem.Eur. J.
2012, 18, 1231−1239.
(10) Seo, Y. J.; Hwang, G. T.; Ordoukhanian, P.; Romesberg, F. E. J.
Am. Chem. Soc. 2009, 131, 3246−3252.
(11) Malyshev, D. A.; Seo, Y. J.; Ordoukhanian, P.; Romesberg, F. E.
J. Am. Chem. Soc. 2009, 131, 14620−14621.
(12) Matsuda, S.; Leconte, A. M.; Romesberg, F. E. J. Am. Chem. Soc.
2007, 129, 5551−5557.
(13) Yu, C.; Henry, A. A.; Romesberg, F. E.; Schultz, P. G. Angew.
Chem., Int. Ed. 2002, 41, 3841−3844.
(14) Seo, Y. J.; Romesberg, F. E. ChemBioChem 2009, 10, 2394−
2400.
(15) Hwang, G. T.; Leconte, A. M.; Romesberg, F. E. ChemBioChem
2007, 8, 1606−1611.
(16) Hari, Y.; Hwang, G. T.; Leconte, A. M.; Joubert, N.; Hocek, M.;
Romesberg, F. E. ChemBioChem 2008, 9, 2796−2799.
(17) Malyshev, D. A.; Pfaff, D. A.; Ippoliti, S. I.; Hwang, G. T.;
Dwyer, T. J.; Romesberg, F. E. Chem.Eur. J. 2010, 16, 12650−
12659.
(18) Stambasky, J.; Hocek, M.; Kocovsky, P. Chem. Rev. 2009, 109,
6729−6764.
(19) Tokuyama, H.; Sato, M.; Ueda, T.; Fukuyama, T. Heterocycles
2001, 54, 105−108.
(20) Ludwig, J.; Eckstein, F. J. Org. Chem. 1989, 54, 631−635.
(21) Knauber, T.; Arikan, F.; Roschenthaler, G. V.; Goossen, L. J.
Chem.Eur. J. 2011, 17, 2689−2697.
(22) Molander, G. A.; Brown, A. R. J. Org. Chem. 2006, 71, 9681−
9686.
(23) Schumann, H.; Kaufmann, J.; Schmalz, H. G.; Bottcher, A.;
Gotov, B. Synlett 2003, 1783−1788.
ASSOCIATED CONTENT
■
(24) Alacid, E.; Najera, C. J. Org. Chem. 2008, 73, 2315−2322.
(25) Farina, V.; Kapadia, S.; Krishnan, B.; Wang, C. J.; Liebeskind, L.
S. J. Org. Chem. 1994, 59, 5905−5911.
S
* Supporting Information
Synthetic methods and compound characterization, pre-steady-
state kinetic assay and data, PCR assay and sequencing data,
calculation of PCR fidelity, and analysis of correlation between
incorporation efficiency and PCR fidelity. This material is
available free of charge via the Internet at http://pubs.acs.org.
(26) Velmathi, S.; Leadbeater, N. E. Tetrahedron Lett. 2008, 49,
4693−4694.
(27) Hocek, M.; Pohl, R.; Klepetar
2005, 4525−4528.
́ ̌ ́
ova, B. Eur. J. Org. Chem. 2005,
5418
dx.doi.org/10.1021/ja312148q | J. Am. Chem. Soc. 2013, 135, 5408−5419

Journal of the American Chemical Society
Article
(28) Joubert, N.; Urban, M.; Pohl, R.; Hocek, M. Synthesis 2008,
1918−1932.
(29) Urban, M.; Pohl, R.; Klepetar
2006, 71, 7322−7328.
(30) Joubert, N.; Pohl, R.; Klepetar
2007, 72, 6797−6805.
(31) Stefko, M.; Slavetinska, L.; Klepetar
Chem. 2011, 76, 6619−6635.
́
o
̌
va,
́
B.; Hocek, M. J. Org. Chem.
́
o
̌
va,
́
B.; Hocek, M. J. Org. Chem.
́ ̌ ́
ova, B.; Hocek, M. J. Org.
(32) Hocek, M.; Fojta, M. Org. Biomol. Chem. 2008, 6, 2233−2241.
(33) Kuchta, R. D.; Mizrahi, V.; Benkovic, P. A.; Johnson, K. A.;
Benkovic, S. J. Biochemistry 1987, 26, 8410−8417.
(34) Wolter, M.; Nordmann, G.; Job, G. E.; Buchwald, S. L. Org. Lett.
2002, 4, 973−976.
(35) Yi, C. Y.; Blum, C.; Lehmann, M.; Keller, S.; Liu, S. X.; Frei, G.;
Neels, A.; Hauser, J.; Schurch, S.; Decurtins, S. J. Org. Chem. 2010, 75,
3350−3357.
(36) Filee, J.; Forterre, P.; Sen-Lin, T.; Laurent, J. J. Mol. Evol. 2002,
54, 763−773.
(37) Kornberg, A.; Baker, T. A. DNA Replication; University Science
Books: Sausalito, CA, 2005.
(38) Chiaramonte, M.; Moore, C. L.; Kincaid, K.; Kuchta, R. D.
Biochemistry 2003, 42, 10472−10481.
(39) Kincaid, K.; Beckman, J.; Zivkovic, A.; Halcomb, R. L.; Engels, J.
W.; Kuchta, R. D. Nucleic Acids Res. 2005, 33, 2620−2628.
(40) Hashimoto, H.; Nishioka, M.; Fujiwara, S.; Takagi, M.; Imanaka,
T.; Inoue, T.; Kai, Y. J. Mol. Biol. 2001, 306, 469−477.
(41) Takagi, M.; Nishioka, M.; Kakihara, H.; Kitabayashi, M.; Inoue,
H.; Kawakami, B.; Oka, M.; Imanaka, T. Appl. Environ. Microbiol. 1997,
63, 4504−4510.
(42) Zhang, X.; Lee, I.; Zhou, X.; Berdis, A. J. J. Am. Chem. Soc. 2006,
128, 143−149.
(43) Zhang, X.; Motea, E.; Lee, I.; Berdis, A. J. Biochemistry 2010, 49,
3009−3023.
(44) Zimmermann, J.; Thielges, M. C.; Seo, Y. J.; Dawson, P. E.;
Romesberg, F. E. Angew. Chem., Int. Ed. 2011, 50, 8333−8337.
(45) Oh, K. I.; Lee, J. H.; Joo, C.; Han, H.; Cho, M. J. Phys. Chem. B
2008, 112, 10352−10357.
(46) Omumi, A.; Beach, D. G.; Baker, M.; Gabryelski, W.;
Manderville, R. A. J. Am. Chem. Soc. 2010, 133, 42−50.
(47) Weisbrod, S. H.; Marx, A. Chem. Commun. 2008, 5675−5685.
(48) Hong, V.; Presolski, S. I.; Ma, C.; Finn, M. G. Angew. Chem., Int.
Ed. 2009, 48, 9879−9883.
5419
dx.doi.org/10.1021/ja312148q | J. Am. Chem. Soc. 2013, 135, 5408−5419