DNA-templated polymerization of side-chain-functionalized peptide nucleic acid aldehydes

Article abstract of DOI:10.1021/ja0753997

The DNA-templated polymerization of synthetic building blocks provides a potential route to the laboratory evolution of sequence-defined polymers with structures and properties not necessarily limited to those of natural biopolymers. We previously reported the efficient and sequence-specific DNA-templated polymerization of peptide nucleic acid (PNA) aldehydes. Here, we report the enzyme-free, DNA-templated polymerization of side-chain- functionalized PNA tetramer and pentamer aldehydes. We observed that polymerization of tetramer and pentamer PNA building blocks with a single lysine-based side chain at various positions in the building block could proceed efficiently and sequence specifically. In addition, DNA-templated polymerization also proceeded efficiently and in a sequence-specific manner with pentamer PNA aldehydes containing two or three lysine side chains in a single building block to generate more densely functionalized polymers. To further our understanding of side-chain compatibility and expand the capabilities of this system, we also examined the polymerization efficiencies of 20 pentamer building blocks each containing one of five different side-chain groups and four different side-chain regio- and stereochemistries. Polymerization reactions were efficient for all five different side-chain groups and for three of the four combinations of side-chain regio- and stereochemistries. Differences in the efficiency and initial rate of polymerization correlate with the apparent melting temperature of each building block, which is dependent on side-chain regio- and stereochemistry but relatively insensitive to side-chain structure among the substrates tested. Our findings represent a significant step toward the evolution of sequence-defined synthetic polymers and also demonstrate that enzyme-free nucleic acid-templated polymerization can occur efficiently using substrates with a wide range of side-chain structures, functionalization positions within each building block, and functionalization densities.

Full text of DOI:10.1021/ja0753997

Published on Web 03/15/2008
DNA-Templated Polymerization of Side-Chain-Functionalized
Peptide Nucleic Acid Aldehydes
Ralph E. Kleiner, Yevgeny Brudno, Michael E. Birnbaum, and David R. Liu*
Contribution from the Howard Hughes Medical Institute and the Department of Chemistry and
Chemical Biology, HarVard UniVersity, Cambridge, Massachusetts 02138
Received July 19, 2007; E-mail: drliu@fas.harvard.edu
Abstract: The DNA-templated polymerization of synthetic building blocks provides a potential route to the
laboratory evolution of sequence-defined polymers with structures and properties not necessarily limited
to those of natural biopolymers. We previously reported the efficient and sequence-specific DNA-templated
polymerization of peptide nucleic acid (PNA) aldehydes. Here, we report the enzyme-free, DNA-templated
polymerization of side-chain-functionalized PNA tetramer and pentamer aldehydes. We observed that
polymerization of tetramer and pentamer PNA building blocks with a single lysine-based side chain at
various positions in the building block could proceed efficiently and sequence specifically. In addition, DNA-
templated polymerization also proceeded efficiently and in a sequence-specific manner with pentamer PNA
aldehydes containing two or three lysine side chains in a single building block to generate more densely
functionalized polymers. To further our understanding of side-chain compatibility and expand the capabilities
of this system, we also examined the polymerization efficiencies of 20 pentamer building blocks each
containing one of five different side-chain groups and four different side-chain regio- and stereochemistries.
Polymerization reactions were efficient for all five different side-chain groups and for three of the four
combinations of side-chain regio- and stereochemistries. Differences in the efficiency and initial rate of
polymerization correlate with the apparent melting temperature of each building block, which is dependent
on side-chain regio- and stereochemistry but relatively insensitive to side-chain structure among the
substrates tested. Our findings represent a significant step toward the evolution of sequence-defined
synthetic polymers and also demonstrate that enzyme-free nucleic acid-templated polymerization can occur
efficiently using substrates with a wide range of side-chain structures, functionalization positions within
each building block, and functionalization densities.
Introduction
polymerization of synthetic building blocks raises the possibility
of performing test tube evolution on synthetic polymers toward
The nucleic acid-templated polymerizations of DNA, RNA,
and proteins1-3 are essential components of all known living
systems. Template-directed polymerization mediates the transla-
tion of genetic information into functional macromolecules and
ultimately enables the evolution of biopolymers. Over the past
few decades, several researchers have explored the use of nucleic
acid-templated polymerization reactions in the absence of
enzymes to generate sequence-defined synthetic oligomers and
polymers without the structural constraints imposed by the
biosynthetic machinery.4-16 The DNA- or RNA-templated
desired functional properties through iterated cycles of transla-
tion (polymerization), selection, amplification, and mutation (as
an example, see Figure 1).17
Building on the findings of Orgel, Lynn, and their respective
co-workers,5,12,14 we previously developed the efficient and
sequence-specific DNA-templated polymerization of unfunc-
tionalized peptide nucleic acid (PNA) aldehydes using reductive
amination coupling reactions.18 In contrast to the 2-methylimi-
dazole-activated phosphate-coupling chemistry pioneered by
Orgel and further developed by other researchers,10,19,20 this
(1) Kuchta, R. D.; Benkovic, P.; Benkovic, S. J. Biochemistry 1988, 27, 6716-
6725.
(11) Kozlov, I. A.; De Bouvere, B.; Van Aerschot, A.; Herdewijn, P.; Orgel, L.
E. J. Am. Chem. Soc. 1999, 121, 2653-2656.
(2) Steitz, T. A. HarVey Lect. 1997-1998, 93, 75-93.
(3) Yonath, A. Annu. ReV. Biophys. Biomol. Struct. 2002, 31, 257-273.
(4) Bag, B. G.; von Keidrowski, G. Pure Appl. Chem 1996, 68, 2145-2152.
(5) Bo¨hler, C.; Nielsen, P. E.; Orgel, L. E. Nature 1995, 376, 578-581.
(6) Chung, N. M.; Lohrmann, R.; Orgel, L. E. Biochim. Biophys. Acta 1971,
228, 536-543.
(12) Li, X.; Zhan, Z.-Y. J.; Knipe, R.; Lynn, D. G. J. Am. Chem. Soc. 2002,
124, 746-747.
(13) Orgel, L. E. Nature 1992, 358, 203-209.
(14) Schmidt, J. G.; Christensen, L.; Nielsen, P. E.; Orgel, L. E. Nucleic Acids
Res. 1997, 25, 4792-4796.
(7) Kozlov, I. A.; Zielinski, M.; Allart, B.; Kerremans, L.; Van, Aerschot, A.;
Busson, R.; Herdewijn, P.; Orgel, L. E. Chem. Eur. J. 2000, 6, 151-155.
(8) Fujimoto, K.; Matsuda, S.; Takahashi, N.; Saito, I. J. Am. Chem. Soc. 2000,
122, 5646-5647.
(15) Schmidt, J. G.; Nielsen, P. E.; Orgel, L. E. Nucleic Acids Res. 1997, 25,
4797-4802.
(16) Walder, J. A.; Walder, R. Y.; Heller, M. J.; Freier, S.; Letsinger, R. L.;
Klotz, I. M. Proc. Natl. Acad. Sci. U.S.A. 1979, 76, 51-55.
(17) Ichida, J. K.; Zou, K.; Horhota, A.; Yu, B.; McLaughlin, L. W.; Szostak,
J. W. J. Am. Chem. Soc. 2005, 127, 2802-2803.
(9) Gat, Y.; Lynn, D. G. In Templated Organic Synthesis; Diederich, F., Stang,
P., Eds.; Wiley: New York, 2000; pp 133-157.
(10) Joyce, G. F. In Cold Spring Harbor Symposium on QuantitatiVe Biology;
Cold Spring Harbor Laboratory Press: Plainview, NY, 1987; Vol. LII, pp
41-51.
(18) Rosenbaum, D. M.; Liu, D. R. J. Am. Chem. Soc. 2003, 125, 13924-
13925.
(19) Inoue, T.; Orgel, L. E. J. Mol. Biol. 1982, 162, 201-217.
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J. AM. CHEM. SOC. 2008, 130, 4646-4659
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DNA-Templated Polymerization
A R T I C L E S
Figure 1. Example of a simplified scheme for the evolution of synthetic polymers using DNA-templated polymerization. (Note that the functional selection
of a DNA-templated polymer will additionally require displacement of the polymer from its DNA template to allow the polymer to fold.)
targets for the nucleic acid-templated polymerization of non-
natural polymers because of their ability to bind DNA sequence
specifically,21 synthetic accessibility, and ability to display side-
chain functional groups that can be derived from corresponding
D- or L-amino acids.22,23 Although functionalized PNA oligomers
containing side-chain modifications have been shown to hybrid-
ize to DNA,22-25 the nucleic acid-templated polymerization of
side-chain-containing building blocks has not been previously
reported despite the potential of functionalized synthetic poly-
mers to possess properties not available to their nonfunction-
alized counterparts.26
In this work we examined the DNA-templated polymerization
of PNA aldehyde tetramer or pentamer building blocks bearing
one or multiple side chains at various positions within the
building block. We observed efficient and sequence-specific
polymerization for building blocks functionalized at any of the
nucleotide positions tested (all but the final position bearing
the aldehyde group) as well as for multiply functionalized
substrates containing up to three lysine side chains. We also
evaluated the DNA-templated polymerization behavior of 20
different PNA aldehyde pentamer building blocks containing
all possible combinations of five side-chain groups and four
side-chain regio- and stereochemistries. Our results show that
three of the four combinations of regio- and stereochemistry
support efficient and sequence-specific DNA-templated polym-
erization, laying the foundation for the use of functionalized
PNA-based building blocks in future templated polymerization-
based synthetic polymer evolution efforts. These findings also
Figure 2. DNA-templated polymerization of unfunctionalized PNA pen-
demonstrate that the nonenzymatic nucleic acid-templated
tamer aldehydes. Reactions were performed at 40 °C in 10 mM TAPS pH
8.5 with 100 mM NaCl using 0.4 µM DNA template and 12.8 µM (4 equiv
(20) Inoue, T.; Orgel, L. E. Science 1983, 219, 859-862.
(21) Nielsen, P. E.; Egholm, M.; Be¨rg, R. H.; Buchardt, O. Science 1991, 254,
1497-1500.
per template codon) of PNA pentamer aldehyde NH2-ggatt-CHO. Prior to
addition of NaBH3CN, reactions were pre-equilibrated to 40 °C by heating
to 95 °C for 10 min and slowly cooling to 40 °C over 15 min. After the
indicated times, the reactions were quenched with excess allyl amine and
subjected to gel filtration and denaturing PAGE analysis (bottom).
(22) Haaima, G.; Lohse, A.; Buchardt, O.; Nielsen, P. E. Angew. Chem., Int.
Ed. 1996, 35, 1939-1942.
(23) Pu¨schl, A.; Sforza, S.; Haaima, G.; Dahl, O.; Nielsen, P. E. Tetrahedron
Lett. 1998, 39, 4707-4710.
(24) Englund, E. A.; Appella, D. H. Org. Lett. 2005, 7, 3465-3467.
(25) Dragulescu-Andrasi, A.; Rapireddy, S.; Frezza, B. M.; Gayathri, C.; Gil,
R. R.; Ly, D. H. J. Am. Chem. Soc. 2006, 128, 10258-10267.
(26) Koppelhus, U.; Awasthi, S. K.; Zachar, V.; Holst, H. U.; Ebbesen, P.;
Nielsen, P. E. Antisense Nucleic Acid Drug DeV. 2002, 12, 51-63.
system relies on typically more efficient DNA-templated
reductive amination reactions first reported by Lynn and co-
workers.12 We considered PNA-based structures ideal initial
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A R T I C L E S
Kleiner et al.
Scheme 1. Synthesis of γ-Functionalized Adenine and Guanine PNA Monomersa
a Route adapted from Englund et al.24 and Thomson et al.45
synthesis of polymers with non-nucleic acid backbones can
support a diverse collection of side-chain structures, attachment
points, stereochemistries, and densitiessfeatures of possible
relevance to prebiotic chemistry.
We prepared DNA templates containing a 5′-amine-termi-
nated hairpin followed by 10 successive repeats of the four-
base DNA codon complementary to both sets of tetramer
building blocks (5′-AACC-3′ for H2N-ggtt-CHO and H2N-ggt*t-
CHO; 5′-ACCC-3′ for H2N-gggt-CHO and H2N-ggg*t-CHO)
(Figure S1a). To evaluate the basic sequence specificity of DNA-
templated polymerization reactions, we also synthesized mis-
matched DNA templates in which five repeats of the appro-
priate matched codon were followed by five repeats of a
mismatched codon (5′-ACAC-3′ for H2N-ggtt-CHO and H2N-
ggt*t-CHO; 5′-CACA-3′ for H2N-gggt-CHO and H2N-ggg*t-
CHO) (Figure S1a). If polymerization proceeds sequence
specifically through a DNA-templated mechanism, as opposed
to a nontemplated, intermolecular mechanism, truncated prod-
ucts ending after five reductive amination couplings should
predominantly result from the use of this mismatched tem-
plate.
Results and Discussion
DNA-Templated Polymerizations of Functionalized PNA
Tetramer Building Blocks. We previously reported the DNA-
templated polymerization of the unfunctionalized NH2-gvvt-
CHO set of PNA aldehyde tetramers (lower case indicates PNA,
v ) a, c, or g).18 On the basis of these findings, we examined
the DNA-templated polymerization of side-chain-functionalized
PNA aldehyde tetramer building blocks. Recent reports have
demonstrated that γ-substituted PNA thymine monomers based
on L-lysine can be readily synthesized and incorporated into
PNA oligomers without impairing DNA hybridization.24 The
γ-substituted L-lysine thymine monomer (t*) was synthesized
following literature precedent24 and incorporated at the third
position of a tetramer PNA aldehyde H2N-ggt*t-CHO using
Fmoc solid-phase peptide synthesis on threonine-linked resin
as we described previously.18 In addition, the nonfunctionalized
PNA tetramer building block H2N-ggtt-CHO was synthesized
for comparison purposes (Table S1). Although this tetramer was
not present in the gvvt set,18 our sequence choice was motivated
by the synthetic accessibility of the functionalized thymine
monomer and the necessity for thymine at the terminal (alde-
hyde) position.
We also constructed a functionalized tetramer aldehyde from
the H2N-gvvt-CHO set by synthesizing a γ-substituted L-lysine-
based guanine PNA monomer. We coupled glycine allyl ester
with Fmoc-lysinal to form the γ-substituted PNA backbone
(Scheme 1). N-Benzhydryloxycarbonyl (Bhoc)-protected guanine-
9-acetic acid was prepared using the method of Debaene and
Winssinger,27 and acylation with the γ-substituted PNA back-
bone using BOP reagent followed by ester deprotection provided
the Fmoc-protected lysine-functionalized PNA guanine mono-
mer acid. The γ-L-lysine-functionalized guanine monomer (g*)
was then used to synthesize PNA aldehyde H2N-ggg*t-CHO
(Table S1).18 We previously showed that the unfunctionalized
H2N-gggt-CHO building block undergoes efficient and sequence-
specific DNA-templated polymerization.18
Polymerization reactions for the functionalized and unfunc-
tionalized tetramer PNA aldehyde building blocks were per-
formed for 30 min at a reaction temperature that balanced
coupling efficiency and sequence specificity (40 °C for H2N-
ggtt-CHO and H2N-ggt*t-CHO; 50 °C for H2N-gggt-CHO and
H2N-ggg*t-CHO) and quenched by addition of excess ally-
lamine. Denaturing PAGE analysis was used to determine the
extent of polymerization for each PNA aldehyde tetramer
(Figures S1a, S1b). The unfunctionalized H2N-ggtt-CHO build-
ing block polymerized efficiently, affording full-length product
in 82% yield (Figures S1a, S1b). Gratifyingly, the L-lysine-
functionalized H2N-ggt*t-CHO tetramer participated in DNA-
templated polymerization with comparable efficiency, providing
full-length product in 90% yield (Figures S1a, S1b). Both the
functionalized and unfunctionalized building block gave pre-
dominantly truncated product when reacted with the mismatched
template (Figure S1a). Polymerization of the H2N-gggt-CHO
and H2N-ggg*t-CHO tetramer building blocks also proceeded
in a highly efficient manner, both affording >80% full-length
40-mer PNA product (Figure S1b) and generating predominantly
truncated product when a mismatched template was used (Figure
S1a). These results demonstrate the ability of the DNA-
templated polymerization of tetramer PNA aldehydes to support
side-chain-functionalized substrates without compromising po-
lymerization efficiency and basic sequence specificity.
(27) Debaene, F.; Winssinger, N. Org. Lett. 2003, 5, 4445-4447.
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PNA Pentamer Building Blocks Containing Lysine Side
Chains at Different Positions. To expand the number of viable
codons with uniform purine content and therefore uniform
template melting temperatures28 in a future PNA selection
system involving side-chain-functionalized building blocks and
mitigate the effect of a more diverse set of potentially
destabilizing side-chain modifications, we turned to pentamer
PNA aldehydes that offer larger codon diversity and greater
potential affinities to DNA templates. We focused our studies
on the PNA pentamer aldehyde H2N-ggatt-CHO. A γ-substituted
L-lysine-based PNA adenine monomer (a*) was prepared
similarly to the g* monomer (Scheme 1). To study the effect
of functionalized monomer position on polymerization, g*, a*,
and t* γ-L-lysine-based monomers were incorporated at the first,
second, third, and fourth positions of the H2N-ggatt-CHO
building block, resulting in H2N-g*gatt-CHO, H2N-gg*att-CHO,
H2N-gga*tt-CHO, and H2N-ggat*t-CHO pentamer PNA alde-
hydes (Figure 3a, Table S1). The terminal, aldehyde-bearing
position was not as readily available for functionalization since
its synthesis involves allylamine,18 which is incompatible with
the Fmoc-protected amino aldehydes used to make the func-
tionalized PNA backbone (data not shown). We also combined
functionalized γ-L-lysine-based monomers in a single building
block to create the multiply functionalized H2N-gg*at*t-CHO,
H2N-gg*a*t*t-CHO, and H2N-g*g*a*t*t-CHO pentamer alde-
hydes (Figure 3a, Table S1) containing two, three, or four side-
chain modifications, respectively.
was used to determine the extent of polymerization for each
PNA aldehyde pentamer (Figure 3a-c).
Consistent with our previous studies using PNA aldehyde
tetramers,18 the unfunctionalized H2N-ggatt-CHO pentamer
polymerized efficiently in a DNA-templated format (Figure 2),
resulting in 85% yield of full-length 40-mer product (confirmed
by MALDI-TOF mass spectrometry) resulting from eight
consecutive coupling reactions (Figure 3c and Supporting
Information, Table S2). In contrast, predominantly truncated
product corresponding to four templated coupling reactions
without additional nontemplated couplings resulted from po-
lymerization reactions conducted under the same conditions but
using the mismatched template (Figure 3b and Supporting
Information, Table S2). These results demonstrate the efficiency
and basic sequence specificity of the DNA-templated unfunc-
tionalized PNA pentamer polymerization system.
Next, we evaluated the polymerization efficiency and se-
quence specificity of the H2N-g*gatt-CHO, H2N-gg*att-CHO,
H2N-gga*tt-CHO, and H2N-ggat*t-CHO PNA building blocks
(Figure 3a) containing γ-L-lysine side chains. The singly
functionalized substrates all polymerized efficiently on DNA
templates, providing full-length products in 76-86% yield
(Figure 3c), similar to that of the unfunctionalized substrate.
The position of the functionalized monomer within the building
block did not affect polymerization efficiency, with the excep-
tion of the H2N-g*gatt-CHO building block, which showed a
slight reduction (∼10%) in polymerization efficiency compared
with the unmodified H2N-ggatt-CHO building block (Figure 3c),
possibly due to the proximity of the functionalized monomer
to the reacting amine group. All of the singly functionalized
building blocks exhibited excellent sequence specificity when
polymerized using the mismatched template (Figure 3a).
We prepared DNA templates containing a 5′-amine-termi-
nated hairpin followed by eight successive repeats of the five-
base DNA codon 5′-AATCC-3′ complementary to the pentamer
PNA building blocks described above (Figure 2). Full-length
polymerization products therefore contain 40 PNA nucleotides
arising from eight consecutive reductive amination coupling
reactions. To evaluate the basic sequence specificity of DNA-
templated polymerization reactions, we also synthesized mis-
matched DNA templates in which four repeats of 5′-AATCC-
3′ were followed by four repeats of the mismatched codon 5′-
ATACC-3′ (Figure 3a). Sequence-specific polymerization using
this mismatched template should result in predominantly trun-
cated products ending after four reductive amination couplings.
The ability of the DNA-templated polymerization to tolerate
side chains at four different nucleotide positions within the
building block raises the possibility that more densely func-
tionalized polymers could be generated using the H2N-gg*at*t-
CHO, H2N-gg*a*t*t-CHO, and H2N-g*g*a*t*t-CHO substrates.
Indeed, the doubly and triply functionalized H2N-gg*at*t-CHO
and H2N-gg*a*t*t-CHO building blocks both polymerized
efficiently, affording full-length product (confirmed by MALDI-
TOF mass spectrometry) in 80% and 70% yield, respectively,
and generating predominantly truncated products in the presence
of the mismatched template (Figure 3a and c; Supporting
Information, Table S2). These results show that the position of
the functionalized monomer has little effect on the polymeri-
zation efficiency and demonstrate the system’s ability to
polymerize highly functionalized building blocks. Although we
were able to observe complete consumption of the DNA
template in the polymerization reaction of the quadruply
functionalized H2N-g*g*a*t*t-CHO building block, we did not
observe a well-defined product species by PAGE analysis. We
speculate that the combination of the highly positively charged
H2N-g*g*a*t*t-CHO building block containing four lysine side
chains and the negatively charged DNA template result in
polymerization product with modest net charge and therefore
poor electrophoretic mobility.
DNA-Templated Polymerizations of Lysine-Containing
PNA Pentamer Building Blocks. Polymerization reactions
contained 0.4 µM DNA template and 12.8 µM PNA building
block (4 equiv per template codon) in aqueous pH 8.5 buffer.
This reaction stoichiometry was determined empirically so as
to balance coupling efficiency and sequence specificity based
upon our previous studies with PNA tetramer aldehydes18 as
well as polymerization experiments with the unfunctionalized
H2N-ggatt-CHO pentamer aldehyde (Figure S2). A solution
containing DNA template and PNA aldehyde was equilibrated
at the reaction temperature (60 °C for H2N-gg*a*t*t-CHO;
55 °C for H2N-gg*at*t-CHO; 50 °C for all other building blocks
containing γ-L-side chains, chosen empirically to balance
coupling efficiency and sequence specificity) before 80 mM
NaBH3CN was added to initiate reductive amination. After 30
min at the reaction temperature, the reactions were quenched
by addition of excess allylamine. Denaturing PAGE analysis
PNA Building Blocks Containing 20 Combinations of
Side-Chain Structure, Stereochemistry, and Regiochemistry.
We also examined the compatibility of DNA-templated polym-
erization with functionalized building blocks bearing side chains
(28) Giesen, U.; Kleider, W.; Berding, C.; Geiger, A.; Orum, H.; Nielsen, P.
Nucleic Acids Res. 1998, 26, 5004-5006.
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A R T I C L E S
Figure 3. (a) Polymerization of functionalized H2N-ggatt-CHO PNA pentamer aldehydes containing one or more γ-L-lysine side chains at various nucleotide
positions in the presence of matched and mismatched DNA templates. Reactions were performed for 30 min at 50 (for singly functionalized building
blocks), 55 (for H2N-gg*at*t-CHO), or 60 °C (for H2N-gg*a*t*t-CHO) in 10 mM TAPS pH 8.5 with 100 mM NaCl using 0.4 µM DNA template and 12.8
µM of the indicated PNA pentamer aldehyde and then quenched with excess allyl amine. Prior to addition of NaBH3CN, reactions were pre-equilibrated to
the reaction temperature as described in Figure 2. (b) Polymerization of side-chain-functionalized H2N-ggat*t-CHO PNA pentamer aldehydes in the presence
of matched and mismatched DNA templates. Reactions were performed as in Figure 3a. Reactions with the unfunctionalized and both R-functionalized
building blocks were performed at 40 °C. Reactions with γ-L-functionalized building blocks were performed at 50 °C, and reactions with γ-D-functionalized
building blocks were performed at 40 °C; temperatures were chosen to optimize efficiency and sequence specificity. (c) Comparison of polymerization
efficiencies of functionalized PNA pentamer aldehydes. Product yield was determined by gel densitometry using denaturing PAGE. Values represent the
average and standard deviation of three independent reactions.
of different structure, regiochemistry, and stereochemistry. The
achiral PNA backbone can be functionalized at the R- or
γ-carbons using one of two side-chain stereochemistries at each
position. The effect of side-chain groups on the melting tem-
perature (Tm) of PNA oligomers is known to be dependent on
the nature of the modification and its regio- and stereochem-
istry.22-25,29 To elucidate the effect of side-chain identity and
position on DNA-templated polymerization, thymine-containing
PNA mononucleotides containing all four combinations of side-
chain attachment point (R or γ) and stereochemistry (D or L)
were synthesized from D- or L-lysine, phenylalanine, leucine,
glutamine, and glutamate in accordance with literature prece-
dent.22,24 The R-modified backbones were prepared by coupling
Fmoc-glycinal with the appropriate benzyl or allyl ester-
protected amino acid (Scheme 2), while the γ-modified back-
bones were made by coupling the appropriate Fmoc-protected
amino aldehyde with glycine benzyl ester (Scheme 3). Acylation
with 1-thymine acetic acid followed by ester deprotection
provided the Fmoc-protected functionalized PNA monomer
acids. In total, we synthesized all 20 functionalized PNA
thymine mononucleotides encompassing aliphatic, aromatic,
polar, anionic, and cationic side-chain groups combined with
both possible side-chain regiochemistries and both possible side-
chain stereochemistries.
Each functionalized thymine PNA monomer (t*) was incor-
porated at the fourth position of a pentamer PNA aldehyde H2N-
ggat*t-CHO. We chose to incorporate diverse side-chain
modifications at this position due to the synthetic accessibility
of functionalized thymine PNA monomers.
DNA-Templated Polymerization of PNA Building Blocks
Containing Diverse Side Chains. We examined the DNA-
templated polymerization efficiency and sequence specificity
of the H2N-ggat*t-CHO PNA building blocks (Figure 3b)
containing the 20 side chains at the t* position using the
approach described above. To balance coupling efficiency and
sequence specificity, reactions with building blocks containing
γ-L-side chains were performed at 50 °C. Reactions with all
other pentamer building blocks were performed at 40 °C. The
substrates bearing side chains at the R-position polymerized
efficiently on DNA templates, providing full-length products
in 66-86% yield (Figure 3c), similar to that of the unfunction-
alized substrate. Side-chain composition did not significantly
(29) Englund, E. A.; Appella, D. H. Angew. Chem., Int. Ed. 2007, 46, 1414-
1418.
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Kleiner et al.
Scheme 2. Synthesis of R-Functionalized PNA Monomersa
a Route adapted from Haaima et al.22 and Englund et al.24
Scheme 3. Synthesis of γ-Functionalized PNA Monomersa
a Route adapted from Englund et al.24
influence the yields of full-length product for a given side-chain
regio- and stereochemistry, although the lysine-based building
blocks consistently afforded slightly higher amounts of full-
length product. While both L and D side-chain stereochemistries
allowed for efficient polymerization, R-D building blocks
produced full-length product yields that were, on average, 7%
higher than those observed from their enantiomers. Both R-L
and R-D building blocks generated predominantly truncated
product in the presence of a mismatched template (Figure 3b),
exhibiting comparable sequence specificity to that of the
nonfunctionalized substrate.
In contrast to the efficient polymerization observed with both
enantiomers of all five R-functionalized building blocks, the
DNA-templated polymerization of PNA aldehydes functional-
ized at the γ-position (Figure 3b), under the above conditions,
was highly dependent on side-chain stereochemistry. Building
blocks containing γ-L side chains were very efficient substrates
for templated polymerization and generated full-length products
in 84% average yield (Figure 3c), the highest of the four types
of side chains described in this work. The enantiomeric γ-D
PNA aldehydes, however, were very poor polymerization
substrates, affording full-length products in only 3-14% yield
(Figure 3c). We also performed the polymerization of γ-D-
functionalized building blocks at 25 °C instead of 40 °C, and
while product yields improved (20-50%, data not shown), they
remained significantly lower than those of the other function-
alized building blocks studied here.
Unlike the dramatic effect of stereochemistry on the polym-
erization of γ-functionalized building blocks, γ-side-chain
structure did not significantly influence polymerization ef-
ficiency. Indeed, the polymerization efficiencies of γ-L-func-
tionalized building blocks (Figure 3c) exhibited the least
sensitivity to variations in side-chain structure of all four types
of side chains tested. In addition, the DNA-templated polym-
9
4652 J. AM. CHEM. SOC. VOL. 130, NO. 14, 2008

DNA-Templated Polymerization
A R T I C L E S
Table 1. Apparent Aggregate Melting Temperature for PNA-DNA
Duplexesa
R
-building block
apparent Tm
(°
C)
γ-building block
apparent Tm (°C)
L-lysine
L-phenylalanine
L-leucine
L-glutamine
L-glutamate
D-lysine
D-phenylalanine
D-leucine
49.9
41.1
46.0
45.1
43.0
53.4
51.1
49.5
52.7
46.3
53.0
L-lysine
L-phenylalanine
L-leucine
L-glutamine
L-glutamate
D-lysine
D-phenylalanine
D-leucine
58.0
59.3
61.0
59.3
54.7
31.1
27.8
30.4
31.5
29.5
D-glutamine
D-glutamate
unfunctionalized
D-glutamine
D-glutamate
a Conditions for Tm measurement: 100 mM NaCl, 10 mM phosphate
buffer, pH 7.2, 0.67 µM (AATCC)8 DNA template, 5.33 µM PNA NH2-
ggat*t-CHO building block. UV measured at 260 nm from 20 to 90 °C in
0.1 °C increments. Values for γ-D-glutamine and γ-D-glutamate correspond
to the major hypochromicity transition (see main text).
erization of γ-functionalized PNA aldehydes exhibited the same
inability to generate significant full-length product in the
presence of mismatched templates as with the R-building blocks
(Figure 3b), although the γ-L building blocks required polym-
erization at a higher temperature (50 °C) to exhibit sequence
specificity comparable to that of the R-functionalized building
blocks at 40 °C (Figure 3b). The polymerization efficiency of
the γ-L building blocks on a matched template did not suffer as
a result of the higher temperature (data not shown). Our results
indicate that the presence of a variety of nonpolar, polar, and
charged side chains in all three polymerization-competent
substrate types (R-L, R-D, and γ-L) does not significantly impair
sequence specificity.
Relationship Between Melting Temperature and Polym-
erization Efficiency. We hypothesized that the observed
differences in polymerization efficiency reflect the ability of a
functionalized building block to bind the DNA template rather
than the ability of a substrate to adopt conformations that favor
polymerization once paired with the template. This model
predicts that polymerization efficiencies should correlate strongly
with melting temperatures of the PNA aldehyde building blocks.
To test this hypothesis, we measured the apparent aggregate
melting temperature of 8 equiv (1 equiv per template codon)
of each functionalized building block as well as that of the
unfunctionalized substrate, hybridized to a linear DNA template
containing eight successive repeats of the 5′-AATCC-3′ codon.
All but two building blocks exhibited a single cooperative
melting transition (Figures S3a-d) presumably corresponding
to the assembly of eight pentamer PNA aldehydes on the DNA
template. Both the γ-D-glutamine and the γ-D-glutamate building
blocks exhibited two cooperative melting transitionssa major
transition at ∼32 °C and a minor transition at ∼47 °C. For these
two building blocks, melting temperatures were computed using
the major transition.
Figure 4. Correlation between apparent aggregate melting temperatures
and polymerization efficiencies of functionalized building blocks. The
melting temperatures (diamonds) from Table 1 are superimposed on the
polymerization efficiency data (bars) from Figure 3c.
building blocks showed modest reductions in Tm. These
observations are consistent with previous reports,22-25,29 al-
though only a small number of studies using γ-modified PNAs
have been described. Taken together, these results support the
hypothesis that melting temperatures of side-chain-modified
PNA aldehydes are the major determinant of their polymeri-
zation efficiencies rather than the possibility that specific side-
chain structures or stereochemistries perturb chemical steps
involved in building block coupling without affecting pairing
to the template.
Modeling the Effects of Side Chains on PNA-DNA
Heteroduplex Structure. To understand the effect of side-chain
regio- and stereochemistry on Tm and polymerization efficiency,
we modeled the conformation that the R-L, R-D, γ-L, and γ-D
building blocks containing a glutamine side chain would adopt
when placed in the middle of a PNA-DNA duplex.30 We
optimized side-chain geometry using molecular mechanics and
the Amber99 force field with an explicit solvation model while
fixing the position of the other PNA-DNA heteroduplex atoms.
The results suggest that the slightly increased polymerization
efficiency and Tm observed for R-D building blocks over R-L
building blocks may arise from the slightly lower steric
hindrance imposed by R-side chains of D stereochemistry (Figure
5b) than those of L stereochemistry (Figure 5a) in the template-
substrate complex. Indeed, a single-point energy calculation
predicts that the R-D-glutamine-containing solvated duplex
structure is stabilized compared with the R-L-glutamine-contain-
ing structure (see Supporting Information).
The apparent melting temperatures (Table 1) correlated
strongly with observed DNA-templated polymerization efficien-
cies for virtually all of the 20 building blocks tested (Figure 4).
Similar to the polymerization efficiency trends, side-chain regio-
and stereochemistry were major determinants of Tm, with γ-L
g R-D g R-L . γ-D, while side-chain structure did not strongly
influence melting temperatures. Compared to unmodified PNA,
the γ-L-functionalized PNA building blocks exhibited signifi-
cantly higher melting temperatures while R-functionalized PNA
In contrast to the R-functionalized substrates, the polymeri-
zation efficiencies and melting temperatures observed for γ-L
building blocks differ greatly from their γ-D counterparts. Our
modeling results suggest that the inability of γ-D-functionalized
building blocks to polymerize efficiently on DNA templates and
(30) Eriksson, M.; Nielsen, P. E. Nat. Struct. Biol. 1996, 3, 410-413.
9
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A R T I C L E S
Kleiner et al.
Figure 6. Correlation between initial rate of product formation and apparent
aggregate melting temperature. Initial rates were measured for R-L-glutamine
(green), R-L-lysine (orange), R-D-phenylalanine (yellow), R-D-glutamine
(brown), γ-L-phenylalanine (purple), γ-L-leucine (blue), and unfunctionalized
(red) building blocks by measuring product formation before 10% of the
template was consumed. Reactions were performed as described in Figures
2 and 3. Melting temperatures are from Table 1. The trend line was generated
by computing a linear regression (r2 ) 0.89) for the data.
Figure 5. PNA-DNA heteroduplex gctat*gtc‚d(GACATAGC) with
glutamine side chain modeled at R- and γ-carbons on the t* thymine PNA
nucleotide: (a) R-L-glutamine modification; (b) R-D-glutamine modification;
(c) γ-L-glutamine modification; (d) γ-L-glutamine modification. Side-chain
geometry was optimized using molecular mechanics with an Amber99 force
field and an explicit solvation model; the other atoms within the PNA-
DNA heteroduplex were fixed during the optimization. Optimization was
performed using HyperChem 8.0, and the optimized structures were rendered
with PyMol. Colors indicate the following: PNA (orange), DNA (red),
D-glutamine (blue), L-glutamine (magenta). (c and d) The PNA thymine
nucleobase that introduces a steric clash with the γ-D-glutamine side chain
is shown as an orange space-filling representation.
building block exhibited both the lowest initial rate of product
formation (3.2% conversion/min) and the lowest Tm (45.1 °C).
These results support the hypothesis that building blocks with
higher apparent aggregate melting temperatures polymerize at
a faster initial rate than do building blocks with lower apparent
melting temperatures (Figure 6). Since the polymerization
reactions are equilibrated prior to reductive amination, building
block Tm determines the proportion of building block annealed
to the template. The correlation between polymerization rate
and melting temperature therefore suggests that under the
conditions tested the rate of covalent bond formation exceeds
the rate of building block-template dissociation and that
building block-template hybridization is at least partially rate
determining for full-length product formation. This characteristic
has been previously observed for other DNA-templated
reactions31-33 when rates of bond formation in the base-paired
complex exceed the rate of template-reagent hybridization.
their unusually low Tm is likely due to a severe steric clash
between the γ-D side chain and the interior of the PNA-DNA
heteroduplex (Figure 5d). Consistent with our findings, this steric
clash is predicted to be completely alleviated in the case of the
γ-L-side-chain stereochemistry (Figure 5c), and single-point
energy calculations predict the γ-L-glutamine structure to be
much more stable than the γ-D-glutamine structure (see Sup-
porting Information).
Polymerization Rate. We further hypothesized that the
kinetics of DNA-templated polymerization should be consistent
with the above trends in PNA-DNA duplex stability. To explore
the effect of building block functionalization on polymerization
kinetics, we measured the initial rates of full-length product
formation at 40 °C for a representative set of R-L-, R-D-, and
γ-L-functionalized building blocks as well as for the unfunc-
tionalized building block. The building blocks were polymerized
as before; however, the reactions were quenched with excess
allyl amine at several time points before 10% of the template
had been consumed. The initial rate of full-length product
formation was then estimated by linear regression.
Conclusion
We describe here the DNA-templated polymerization of
functionalized PNA aldehydes, representing the first nonenzy-
matic translation of a nucleic acid sequence into a side-chain-
containing synthetic polymer. Researchers have also been able
to generate modified biopolymers enzymatically by exploiting
the tolerance of DNA34-39 and RNA40-42 polymerases or of the
(31) Gartner, Z. J.; Grubina, R.; Calderone, C. T.; Liu, D. R. Angew. Chem.,
Int. Ed. 2003, 42, 1370-1375.
(32) Gartner, Z. J.; Kanan, M. W.; Liu, D. R. Angew. Chem., Int. Ed. 2002, 41,
1796-1800.
(33) Gartner, Z. J.; Liu, D. R. J. Am. Chem. Soc. 2001, 123, 6961-6963.
(34) Horhota, A.; Zou, K.; Ichida, J. K.; Yu, B.; McLaughlin, L. W.; Szostak,
J. W.; Chaput, J. C. J. Am. Chem. Soc. 2005, 127, 7427-7434.
(35) Latham, J. A.; Johnson, R.; Toole, J. J. Nucleic Acids Res. 1994, 22, 2817-
2822.
At 40 °C, the γ-L building blocks polymerize at the fastest
initial rate (∼13% conversion/min) and have the highest
apparent Tm (∼60 °C). The R-substituted building blocks attain
comparable product yield after 30 min (Figure 3b); however,
they polymerize with a substantially decreased initial rate (3-
7% conversion/min), consistent with their lower Tm’s (45-
53 °C), than the γ-L building blocks. Among the building blocks
whose polymerization rates were measured, the R-L-glutamine
(36) Lee, S. E.; Sidorov, A.; Gourlain, T.; Thorpe, S. J.; Brazier, J. A.; Dickman,
M. J.; Hornby, D. P.; Grasby, J. A.; Williams, D. M. Nucleic Acids Res.
2001, 29, 1565-1573.
(37) Perrin, D. M.; Garestier, T.; Hele`ne, C. J. Am. Chem. Soc. 2001, 123, 1556-
1663.
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2875.
9
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DNA-Templated Polymerization
A R T I C L E S
ribosome43,44 for modified substrates; such an approach, how-
ever, is limited to backbone structures that are compatible with
the biosynthetic machinery. The findings reported here lay a
foundation for the evolution of sequence-defined synthetic
polymers bearing side-chain functionality determined by the
researcher rather than by compatibility with the biosynthetic
machinery. In addition, these results demonstrate that oligomeric
building blocks containing side chains of varying position within
the building block, structure, regiochemistry, and stereochem-
istry can participate in the nonenzymatic translation of nucleic
acid sequences into non-nucleic acid polymers, a step presumed
to have occurred (though under very different conditions than
the ones used here) in the prebiotic world.
We found that PNA aldehydes functionalized with lysine side
chains at various positions within the building block undergo
efficient and sequence-specific DNA-templated polymerization.
The tolerance of this reaction to the position of the nucleotide
containing the side chain within the building block also enables
DNA-templated polymerization to generate densely function-
alized sequence-defined synthetic polymers from multiply
functionalized building blocks, including a PNA 40-mer in
which 24 of the PNA nucleotides contain a side-chain group.
In addition, our studies reveal that polar, positively and
negatively charged, aromatic, and aliphatic side chains can be
polymerized in an efficient and sequence-programmed manner
on a DNA template. By measuring the polymerization efficien-
cies of building blocks modified at the R- and γ-positions with
both possible stereochemistries at each position, we observed
that all functionalized building blocks with the exception of
those with D-side chains at the γ-carbon are efficient DNA-
templated polymerization substrates. Polymerization efficiency
depends predominantly on side-chain position and stereochem-
istry rather than on side-chain size, hydrophobicity, or charge.
Apparent aggregate melting temperatures correlate with polym-
erization rate and suggest that building block hybridization to
the template is at least partially rate limiting for polymerization.
In light of our findings, we conclude that PNA aldehydes
functionalized at the γ-carbon with L-side chains or at the
R-carbon with either side-chain stereochemistry are efficient
participants in DNA-templated polymerization. In contrast, γ-D-
modified PNA building blocks are unsuitable for DNA-
templated polymerization, likely because of poor hybridization
to the template resulting from steric interference with the PNA
nucleobases.
proteins and nucleic acids by virtue of specialized building
blocks containing, for example, biophysical probes, small-
molecule catalysts, electroactive groups, or known ligands to
biological targets.
Experimental Section
General Methods. Small molecules were purified by flash chro-
matography using a Biotage SP4 purification system and a hexanes/
ethyl acetate gradient. Analytical thin layer chromatography (TLC) was
performed on E. Merck precoated (0.25 mm) silica gel 60-F254 plates.
Visualization was accomplished with UV light and staining with 0.3%
ninhydrin in n-butanol followed by heating. Characterization of small-
molecule products was accomplished through proton magnetic reso-
nance (1H NMR) and carbon nuclear magnetic resonance (13C NMR)
obtained using Varian iNOVA 500 (500 MHz) NMR spectrophotom-
eters at 22 °C as well as electrospray mass spectrometry on a Waters
Q-TOF Premier instrument. Unless otherwise noted, all commercially
available reagents and solvents were purchased from Aldrich and used
without further purification. Tetrahydrofuran (THF) was distilled from
sodium and benzophenone prior to use. Dimethylformamide (DMF)
and CH2Cl2 were purified by passage through activated alumina. Fmoc-
protected amino acids were purchased from Novabiochem. Amino ester
hydrochlorides were purchased from Chem-Impex International, Inc.
All nonfunctionalized PNA monomers were purchased from Applied
Biosystems.
DNA oligonucleotides were synthesized using standard automated
solid-phase phosphoramidite coupling methods on a PerSeptive Bio-
systems Expedite 8909 DNA synthesizer. All reagents and phosphora-
midites for DNA synthesis were purchased from Glen Research.
Oligonucleotides were purified by reverse-phase HPLC using a C18
stationary phase and an acetonitrile/100 mM triethyl ammonium acetate
(TEAA) gradient and quantitated using UV spectroscopy carried out
on a Nanodrop ND1000 Spectrophotometer.
Abbreviations. RT, room temperature; Fmoc, fluorenyl methoxy
carbonyl; Boc, tert-butyl oxy carbonyl; BOP, benzotriazole-1-yl-oxy-
tris-(dimethylamino)-phosphonium hexafluorophosphate; DhbtOH, 3-hy-
droxy-1,2,3-benzo-triazin-4(3H)-one; EDC, 1-ethyl-3-(3-dimethylami-
nopropyl) carbodiimide hydrochloride; HOBt, 1-hydroxybenzotriazole;
EtOAc, ethyl acetate; TFA, trifluoroacetic acid; TAPS, N-tris(hy-
droxymethyl)methyl-3-aminopropanesulfonic acid; TBE, tris/borate/
EDTA.
Synthesis of γ-L-Lysine-Functionalized Guanine and Adenine
Fmoc-PNA Monomers. The synthesis of γ-L-lysine-functionalized
Fmoc-PNA guanine and adenine monomers was adapted from the
previously described syntheses of the Boc-γ-L-lysine(Fmoc) thymine
monomer24 and unfunctionalized Fmoc-PNA guanine and adenine
monomers.45
tert-Butyl (S)-5-(((9H-fluoren-9-yl)methoxy)carbonylamino)-6-
oxohexyl-carbamate (1). This compound was prepared according to
the protocols of Wang et al.46
The DNA-templated polymerization of functionalized build-
ing blocks combined with functional selection, amplification,
and template mutation provides a potential method for realizing
the laboratory evolution of sequence-defined polymers bearing
diverse chemical functional groups. Such a system raises the
possibility of discovering synthetic polymers with functional
properties that in principle could extend beyond those of natural
(S)-Allyl 2-(2-(((9H-Fluoren-9-yl)methoxy)carbonylamino)-6-(tert-
butoxycarbonylamino)hexylamino)acetate (2). To a stirring solution
of Boc-Gly-OH (3 g) in 30 mL of dry acetonitrile was added Cs2CO3
(6.70 g). After 30 min, allyl bromide (1.95 g) was added. After stirring
overnight, the reaction mixture was evaporated under reduced pressure.
The resulting residue was suspended in EtOAc and washed with H2O,
1 M aqueous HCl, saturated aqueous NaHCO3, and brine. The organic
layer was dried over MgSO4 and evaporated under reduced pressure
to yield 3.32 g (90.0%) of Boc-Gly-OAll as a yellow oil. Boc-Gly-
OAll (3.32) was dissolved in 20 mL of anhydrous TFA and stirred for
1 h. The reaction was evaporated under reduced pressure, after which
(39) Thum, O.; Ja¨ger, S.; Famulok, M. Angew. Chem., Int. Ed. 2001, 40, 3990-
3993.
(40) Dewey, T. M.; Mundt, A. A.; Crouch, G. J.; Zyzniewski, M. C.; Eaton, B.
E. J. Am. Chem. Soc. 1995, 117, 8474-8475.
(41) Matulic-Adamic, J.; Daniher, A. T.; Karpeisky, A.; Haeberli, P.; Sweedler,
D.; Beigelman, L. Bioorg. Med. Chem. Lett. 2000, 10, 1299-1302.
(42) Vaish, N. K.; Fraley, A. W.; Szostak, J. W.; McLaughlin, L. W. Nucleic
Acids Res. 2000, 28, 3316-3322.
(43) Forster, A. C.; Tan, Z.; Tan, M. N.; Nalam, H.; Lin, H.; Qu, H.; Cornish,
V. W.; Blacklow, S. C. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 6353-
6357.
(45) Thomson, S. A.; Josey, J. A.; Cadilla, R.; Gaul, M. D.; Hassman, C. F.;
Luzzio, M. J.; Pipe, A. J.; Reed, K. L.; Ricca, D. J.; Wiethe, R. W.; Noble,
S. A. Tetrahedron 1995, 51, 6179-6194.
(46) Wang, G.; Mahesh, U.; Chen, G. Y. J.; Yao, S. Q. Org. Lett. 2003, 5,
737-740.
(44) Josephson, K.; Hartman, M. C. T.; Szostak, J. W. J. Am. Chem. Soc. 2005,
127, 11727-11735.
9
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A R T I C L E S
Kleiner et al.
toluene and CH2Cl2 were added and evaporated under reduced pressure
to get rid of residual TFA. The resulting crystalline solid was suspended
in 10 mL of dry CH2Cl2 and dissolved by addition of N,N-diisopro-
pylethylamine (2.0 mL). To this stirring solution was added 1 (1.10 g)
in 10 mL of dry CH2Cl2. After 15 min, sodium triacetoxyborohydride
(621 mg) was added. After 1 h, the reaction was quenched by addition
of 15 mL of saturated aqueous Na2CO3. After stirring for 10 min, the
reaction mixture was extracted with CH2Cl2. The combined organic
layers were dried over MgSO4 and evaporated under reduced pressure.
The resulting oil was purified by flash column chromatography [Rf )
0.50 (EtOAc)] to yield 1.20 g (89.0%) of 2 as a white foam. 1H NMR
(500 MHz, CDCl3): δ 7.69 (d, J ) 10 Hz, 2H, Fmoc-H), 7.56 (d, J
) 10 Hz, 2H, Fmoc-H), 7.29 (m, 4H, Fmoc-H), 5.85 (m, 1H, CH2-
CHdCH2), 5.43 (br s, 1H, Fmoc-NH), 5.17-5.30 (m, 2H, CHdCH2),
4.84 (br s, 1H, Boc-NH), 4.56 (d, J ) 2.5 Hz, 2H, O-CH2-CHd
CH2), 4.36 (d, J ) 5 Hz, 2H, Fmoc-CH-CH2-O), 4.16 (t, J ) 2.5
Hz, 1H, Fmoc-CH-CH2), 3.63 (s, 1H, Fmoc-NH-CH-CH2), 3.34
(m, 2H, NH-CH2-CO2All), 3.04 (d, J ) 10 Hz, 2H, Boc-NH-CH2-
CH2), 2.59 (m, 2H, Fmoc-NH-CH-CH2-NH), 1.2-1.5 (m, 6H,
CH-CH2-CH2-CH2-CH2), 1.43 (s, 9H, tert-butyl-CH3). 13C NMR
(125 MHz, CDCl3): δ 172.4, 156.7, 144.3, 141.5, 132.1, 127.8, 127.2,
125.3, 120.1, 118.8, 79.1, 66.5, 65.6, 60.6, 53.0, 51.1, 50.9, 47.5, 40.4,
32.8, 30.0, 28.7, 23.2, 14.4. HRMS (ESI-MS m/z) mass calcd for
C31H41N3O6 [M + H]+, 552.31; found, 552.31.
CH-CH2-NH), 1.2-1.5 (m, 6H, CH-CH2-CH2-CH2-CH2), 1.43
(s, 9H, tert-butyl-CH3). 13C NMR (125 MHz, DMSO): δ 207.2, 156.5,
154.2, 144.3, 141.5, 140.7, 135.5, 134.4, 133.8, 132.2, 127.2-129.5,
120.8, 78.7, 47.8, 31.4, 29.0, 22.6. LRMS (ESI-MS m/z) mass calcd
for C49H52N8O10 [M - H]-, 911.37; found, 911.3.
2-(6-(Benzhydryloxycarbonylamino)-adenin-9-yl)acetic Acid (3b).
This compound was prepared according to the protocols of Debaene
and Winssinger.27
(S)-Allyl 2-(N-(2-(((9H-Fluoren-9-yl)methoxy)carbonylamino)-6-
(tert-butoxycarbonylamino)hexyl)-2-(6-(benzhydryloxycarbonylami-
no)-adenin-9-yl)acetamido)acetate (4b). Following the procedure used
to prepare compound 4a, 2 (600 mg) was converted to 350 mg (34.3%)
1
of 4b [Rf ) 0.70 (10% MeOH/EtOAc)] as a white solid. H NMR
(500 MHz, CDCl3): δ major rotamer only 9.03 (s, 1H, Bhoc-NH),
8.64 (s, 1H, adenine-H), 7.96 (s, 1H, adenine-H), 7.10-7.69 (m, 18H,
Fmoc-H + phenyl-H), 6.96 (Bhoc-CH), 5.85 (m, 1H, CH2-CHd
CH2), 5.43 (br s, 1H, Fmoc-NH), 5.17-5.30 (m, 2H, CHdCH2), 4.84
(br s, 1H, Boc-NH), 4.56 (d, J ) 2.5 Hz, 2H, O-CH2-CHdCH2),
4.42 (m, 2H, adenine-CH2-CO), 4.36 (d, J ) 5 Hz, 2H, Fmoc-CH-
CH2-O), 4.16 (t, J ) 2.5 Hz, 1H, Fmoc-CH-CH2), 3.63 (s, 1H,
Fmoc-NH-CH-CH2), 3.34 (m, 2H, NH-CH2-CO2All), 3.04 (d, J
) 10 Hz, 2H, Boc-NH-CH2-CH2), 2.59 (m, 2H, Fmoc-NH-CH-
CH2-NH), 1.2-1.5 (m, 6H, CH-CH2-CH2-CH2-CH2), 1.43 (s, 9H,
tert-butyl-CH3). 13C NMR (125 MHz, CDCl3) δ 171.4, 168.8, 167.5,
166.9, 156.7, 152.9, 152.8, 151.7, 150.6, 149.5, 144.3, 141.5, 132.1,
127.8, 127.2, 125.3, 121.5, 120.1, 119.0, 79.4, 78.9, 66.5, 65.6, 60.6,
53.0, 51.1, 50.9, 47.5, 40.4, 32.8, 30.0, 28.7, 23.2, 14.4. LRMS (ESI-
MS m/z) mass calcd for C52H57N8O9 [M + H]+, 937.42; found, 937.4.
(S)-2-(N-(2-(((9H-Fluoren-9-yl)methoxy)carbonylamino)-6-(tert-
butoxycarbonylamino)hexyl)-2-(6-(benzhydryloxycarbonylamino)-
adenin-9-yl)acetamido)acetic Acid (5b). Following the procedure used
to prepare compound 5a, 4b (350 mg) was converted to 269 mg (80.5%)
of 5b as a yellow solid. 1H NMR (500 MHz, DMSO): δ major rotamer
only 10.89 (s, 1H, Bhoc-NH), 8.50 (m, 1H, adenine-H), 7.96 (s, 1H,
adenine-H), 7.10-7.69 (m, 18H, Fmoc-H + phenyl-H), 6.96
(Bhoc-CH), 5.85 (m, 1H, CH2-CHdCH2), 5.43 (br s, 1H, Fmoc-
NH), 4.84 (br s, 1H, Boc-NH), 4.42 (m, 2H, adenine-CH2-CO), 4.36
(d, J ) 5 Hz, 2H, Fmoc-CH-CH2-O), 4.16 (t, J ) 2.5 Hz, 1H,
Fmoc-CH-CH2), 3.63 (s, 1H, Fmoc-NH-CH-CH2), 3.34 (m, 2H,
NH-CH2-CO2H), 3.04 (d, J ) 10 Hz, 2H, Boc-NH-CH2-CH2),
2.59 (m, 2H, Fmoc-NH-CH-CH2-NH), 1.2-1.5 (m, 6H, CH-
CH2-CH2-CH2-CH2), 1.43 (s, 9H, tert-butyl-CH3). 13C NMR (125
MHz, DMSO): δ 171.4, 167.4, 156.9, 153.0, 144.5, 141.6, 132.2,
127.2-129.2, 125.9, 120.8, 77.9, 65.7, 47.5, 45.2, 32.0, 30.0, 29.0,
23.6. LRMS (ESI-MS m/z) mass calcd for C49H52N8O9 [M - H]-,
895.38; found, 895.3.
2-(2-(Benzhydryloxycarbonylamino)-guanin-9-yl)acetic Acid (3a).
This compound was prepared according to the protocols of Debaene
and Winssinger.27
(S)-Allyl 2-(N-(2-(((9H-Fluoren-9-yl)methoxy)carbonylamino)-6-
(tert-butoxycarbonylamino)hexyl)-2-(2-(benzhydryloxycarbonylami-
no)-guanin-9-yl)acetamido)acetate (4a). To a stirring solution of 2
(600 mg) in 8 mL of dry DMF was added 3a (457 mg), BOP reagent
(602 mg), HOBt (184 mg), and N,N-diisopropylethylamine (285 µL).
After 6 h, the reaction was evaporated under reduced pressure. The
resulting residue was dissolved in EtOAc and washed with H2O, 1 M
aqueous HCl, saturated aqueous NaHCO3, and brine. The organic layer
was dried over MgSO4 and evaporated under reduced. The resulting
oil was purified by flash column chromatography [Rf ) 0.40 (10%
1
MeOH/EtOAc)] to yield 386 mg (37.2%) of 11a as a white solid. H
NMR (500 MHz, CDCl3): δ major rotamer only 7.98 (s, 1H, guanine-
H), 7.10-7.80 (m, 18H, Fmoc-H + phenyl-H), 6.36 (s, 1H, Bhoc-
CH-O), 5.85 (m, 1H, CH2-CHdCH2), 5.14-5.33 (m, 2H, CHdCH2),
4.80 (s, 2H, guanine-CH2-CO), 4.54 (d, J ) 2.5 Hz, 2H, O-CH2-
CHdCH2), 4.18-4.42 (m, 4H, guanine-CH2-CO + Fmoc-CH-
CH2-O), 4.16 (t, J ) 2.5 Hz, 1H, Fmoc-CH-CH2), 3.63 (s, 1H,
Fmoc-NH-CH-CH2), 3.34 (m, 2H, NH-CH2-CO2All), 3.04 (d, J
) 10 Hz, 2H, Boc-NH-CH2-CH2), 2.59 (m, 2H, Fmoc-NH-CH-
CH2-NH), 1.2-1.5 (m, 6H, CH-CH2-CH2-CH2-CH2), 1.43 (s, 9H,
tert-butyl-CH3). 13C NMR (125 MHz, CDCl3): δ 168.7, 167.1, 166.8,
162.8, 157.7, 156.7, 149.3, 147.4, 143.9, 141.5, 131.8, 127.2-128.8,
124.6, 120.2, 119.2, 118.8, 78.9, 66.2, 53.0, 50.1, 47.6, 44.4, 40.4, 36.7,
31.7, 28.7, 23.2. HRMS (ESI-MS m/z) mass calcd for C52H57N8O10
[M + H]+, 953.42; found, 953.42.
(S)-2-(N-(2-(((9H-Fluoren-9-yl)methoxy)carbonylamino)-6-(tert-
butoxycarbonylamino)hexyl)-2-(2-(benzhydryloxycarbonylamino)-
guanin-9-yl)acetamido)acetic Acid (5a). To a stirring solution of 4a
(386 mg) in 8 mL of THF at RT was added N-ethyl aniline (960 µL)
followed by (Ph3P)4Pd (205 mg). After stirring for 60 min, the reaction
was evaporated under reduced pressure to give a yellow film. The
yellow film was redissolved in EtOAc and washed with saturated
aqueous NaHSO4, H2O, and brine to afford 250 mg (68%) of 5a as a
yellow solid. 1H NMR (500 MHz, DMSO): δ major rotamer only 11.20
(s, 1H, guanine-H), 7.20-7.90 (m, 18H, Fmoc-H + phenyl-H), 6.84
(s, 1H, Bhoc-CH-O), 4.18-4.42 (m, 4H, guanine-CH2-CO +
Fmoc-CH-CH2-O), 4.16 (t, J ) 2.5 Hz, 1H, Fmoc-CH-CH2), 3.63
(s, 1H, Fmoc-NH-CH-CH2), 3.34 (m, 2H, NH-CH2-CO2H), 3.04
(d, J ) 10 Hz, 2H, Boc-NH-CH2-CH2), 2.59 (m, 2H, Fmoc-NH-
Synthesis of R-Functionalized Thymine Fmoc-Peptide Nucleic
Acid (PNA) Monomers. The synthesis of R-functionalized Fmoc-PNA
thymine monomers was adapted from the previously described syntheses
of R-functionalized Boc-PNA thymine monomers.22,23 The representa-
tive synthesis of the R-L-lysine-thymine monomer is provided below.
(4H-Fluoren-9-yl)methyl 2-oxoethylcarbamate (6). This compound
was prepared according to the protocols of Wang et al.46
(S)-Allyl 2-(2-(((4H-Fluoren-9-yl)methoxy)carbonylamino)ethy-
lamino)-6-(tert-butoxycarbonylamino)hexanoate (7a). To a stirring
solution of 6 (773 mg) and Nꢀ-Boc-L-lysine allyl ester hydrochloride
in 12 mL of dry CH2Cl2 was slowly added N,N-diisopropylethylamine
(340 µL) over 2 min. After stirring for 10 min at RT, sodium
triacetoxyborohydride (525 mg) was added to the reaction. After 90
min of additional stirring at RT, the reaction was quenched with
saturated aqueous Na2CO3. The reaction mixture was extracted with
CH2Cl2, and the combined organic layers were dried over MgSO4 and
evaporated under reduced pressure. The resulting oil was purified by
flash column chromatography [Rf ) 0.60 (EtOAc)] to yield 716 mg
1
(61.2%) of 7a as a clear, colorless oil. H NMR (500 MHz, CDCl3):
δ 7.70 (d, J ) 10 Hz, 2H, Fmoc-H), 7.56 (d, J ) 10 Hz, 2H, Fmoc-
9
4656 J. AM. CHEM. SOC. VOL. 130, NO. 14, 2008

DNA-Templated Polymerization
A R T I C L E S
H), 7.29 (m, 4H, Fmoc-H), 5.86 (m, 1H, CH2-CHdCH2), 5.58 (br s,
1H, Fmoc-NH), 5.17-5.30 (m, 2H, CHdCH2), 4.81 (br s, 1H, Boc-
NH), 4.57 (d, J ) 2.5 Hz, 2H, O-CH2-CHdCH2), 4.34 (d, J ) 5 Hz,
2H, Fmoc-CH-CH2-O) 4.17 (t, J ) 2.5 Hz, 1H, Fmoc-CH-CH2),
3.25 (m, 1H, NH-CH-CO2All), 3.17 (t, 2H, J ) 2.5 Hz, Fmoc-
NH-CH2-CH2), 3.04 (m, 2H, Boc-NH-CH2-CH2), 2.50-2.76 (m,
2H, Fmoc-NH-CH2-CH2-NH), 1.16-1.66 (m, 6H, CH-CH2-
CH2-CH2-CH2), 1.41 (s, 9H, tert-butyl-CH3). 13C NMR (125 MHz,
CDCl3): δ 174.2, 156.3, 155.9, 143.9, 141.0, 131.7, 127.0, 127.6, 124.8,
120.0, 119.2, 78.6, 66.2, 65.1, 60.9, 60.0, 47.0, 40.9, 40.0, 32.8, 29.9,
28.0, 22.4, 14.0. HRMS (ESI-MS m/z) mass calcd for C31H41N3O6 [M
+ H]+, 552.31; found, 552.30.
+ thymine-H + phenyl-H), 6.88 (s, 1H, Fmoc-NH), 4.5-4.6 (m,
1H, N-CH-CO2H) 4.53 (q, J ) 20 Hz, 2H, thymine-CH2-CO), 4.27
(d, J ) 10 Hz, 1H, Fmoc-CH-CH2-O), 4.17 (t, J ) 5 Hz, 2H, Fmoc-
CH-CH2), 2.60-3.20 (m, 6H, phenyl-CH2-CH + Fmoc-NH-CH2-
CH2-N + Fmoc-NH-CH2-CH2), 1.76 (s, 3H, thymine-CH3). 13C
NMR (125 MHz, DMSO): δ 169.1, 168.5, 166.3, 165.2, 165.1, 165.0,
151.8, 151.7, 151.5, 143.2, 143.1, 142.8, 141.5, 140.6, 140.1, 139.0,
138.1, 136.1, 133.2, 130.6, 130.2, 129.7, 129.6, 129.2, 128.9, 128.8,
128.4, 127.8, 127.7, 127.2, 126.5, 124.9, 124.0, 122.1, 120.7, 120.6,
110.5, 109.3, 108.7, 78.9, 43.7, 42.5, 28.1, 18.8, 12.7, 12.6. LRMS
(ESI-MS m/z) mass calcd for C33H32N4O7 [M - H]-, 595.22; found,
595.2.
(S)-2-(N-(2-(((9H-Fluoren-9-yl)methoxy)carbonylamino)ethyl)-2-
(thymin-1(2H)-yl)acetamido)-3-phenylpropanoic Acid. All charac-
terization data matched 9b.
(S)-Allyl 2-(N-(2-(((4H-Fluoren-9-yl)methoxy)carbonylamino)-
ethyl)-2-(thymin-1(2H)-yl) acetamido)-6-(tert-butoxycarbonylami-
no)hexanoate (8a). To a stirring solution of 7a (358 mg), 1-thymine
acetic acid (239 mg), and DhbtOH (212 mg) in DMF (13 mL) at
40 °C was added EDC (268 mg). After stirring overnight at 40 °C,
H2O was then added, forming a white precipitate, and the reaction
mixture was extracted into EtOAc. The combined organic layers were
washed with 1 M aqueous HCl, saturated aqueous NaHCO3, H2O, and
brine. The organic layer was dried over MgSO4 and evaporated under
(R)-2-(N-(2-(((9H-Fluoren-9-yl)methoxy)carbonylamino)ethyl)-2-
(thymin-1(2H)-yl)acetamido)-4-methylpentanoic Acid (9c). 1H NMR
(500 MHz, DMSO): δ 11.28 (s, 1H, imide-NH), 7.86 (m, 2H, Fmoc-
H), 7.67 (m, 2H, Fmoc-H) 7.2-7.5 (m, 5H, Fmoc-H + thymine-
H), 6.86 (s, 1H, Fmoc-NH), 4.65 (s, 2H, thymine-CH2-CO), 4.2-
4.6 (m, 4H, N-CH-CO2H + Fmoc-CH-CH2-O + Fmoc-CH-
CH2), 3.36 (m, 2H, Fmoc-NH-CH2-CH2-N), 3.20 (m, 2H, Fmoc-
NH-CH2-CH2), 1.4-1.8 (m, 3H, CH3-CH-CH3 + CH-CH2-CH),
1.74 (s, 3H, thymine-CH3) 0.86 (m, 6H, CH3-CH-CH3). 13C NMR
(125 MHz, DMSO): δ 173.3, 170.3, 168.4, 165.2, 157.0, 156.3, 152.2,
151.7, 144.5, 143.1, 141.5, 139.9, 136.2, 134.3, 133.2, 131.9, 128.9,
128.8, 128.6, 128.3, 127.7, 125.8, 125.6, 125.0, 120.8, 117.0, 115.2,
108.7, 67.7, 66.1, 57.1, 48.9, 47.5, 45.6, 38.3, 35.1, 31.1, 25.8, 25.1,
23.5, 22.5, 12.6. LRMS (ESI-MS m/z) mass calcd for C30H34N4O7
[M - H]-, 561.23; found, 561.2.
1
reduced pressure to afford 426 mg (93.4%) of 8a as a white solid. H
NMR (500 MHz, DMSO): δ major rotamer 11.33 (s, 1H, imide-NH),
7.86 (d, J ) 10 Hz, 2H, Fmoc-H), 7.67 (d, J ) 10 Hz, 2H, Fmoc-
CH), 7.28-7.42 (m, 5H, Fmoc-H + thymine-H), 6.77 (m, 1H,
Fmoc-NH), 5.87 (m, 1H, CH2-CHdCH2), 5.15-5.31 (m, 2H, CHd
CH2), 4.77 (s, 2H, thymine-CH2-CO), 4.52 (d, J ) 2.5 Hz, 2H,
O-CH2-CHdCH2), 4.37 (d, J ) 5 Hz, 2H, Fmoc-CH-CH2-O),
4.32 (m, 1H, N-CH-CO2All), 4.22 (t, J ) 2.5 Hz, 1H, Fmoc-CH-
CH2), 3.40 (m, 2H, Fmoc-NH-CH2-CH2-NH), 3.29 (m, 2H, Fmoc-
NH-CH2-CH2), 2.91 (m, 2H, Boc-NH-CH2-CH2) 1.75 (s, 3H,
thymine-CH3), 1.10-1.50 (m, 6H, CH-CH2-CH2-CH2-CH2), 1.35
(s, 9H, tert-butyl-CH3), minor rotamer 5.96 (m, 1H, CH2-CHdCH2),
5.20-5.37 (m, 2H, CHdCH2), 1.77 (s, 3H, thymine-CH3). 13C NMR
(125 MHz, DMSO): δ 171.3, 168.2, 165.2, 157.0, 156.4, 151.9, 144.3,
143.0, 141.8, 133.2, 128.4, 127.6, 125.8, 121.0, 118.2, 108.6, 78.0,
66.2, 65.9, 60.4, 49.2, 47.7, 47.0, 38.1, 30.2, 29.0, 23.9, 21.7, 15.3,
12.6. HRMS (ESI-MS m/z) mass calcd for C38H47N5O9 [M + H]+,
718.35; found, 718.34.
(S)-2-(N-(2-(((9H-Fluoren-9-yl)methoxy)carbonylamino)ethyl)-2-
(thymin-1(2H)-yl)acetamido)-4-methylpentanoic Acid. All charac-
terization data matched 9c.
(S)-2-(N-(2-(((9H-Fluoren-9-yl)methoxy)carbonylamino)ethyl)-2-
(thymin-1(2H)-yl)acetamido)-5-oxo-5-(tritylamino)pentanoic Acid
(9d). 1H NMR (500 MHz, DMSO): δ 11.27 (s, 1H, imide-NH), 8.58
(s, 1H, trityl-NH), 7.87 (d, J ) 9.0 Hz, 2H, Fmoc-H), 7.65 (m, 2H,
Fmoc-H), 7.10-7.44 (m, 20H, thymine-H + trityl-H + Fmoc-H),
4.61 (m, 2H, thymine-CH2-CO), 4.33 (d, 2H, J ) 10 Hz, Fmoc-
CH-CH2-O), 4.24 (m, 1H, N-CH-CO2H), 4.14 (t, 1H, J ) 10 Hz,
Fmoc-CH-CH2), 3.58 (m, 2H, Fmoc-NH-CH2-CH2-N), 3.20 (m,
2H, Fmoc-NH-CH2-CH2), 2.00-2.44 (m, 4H, CH-CH2-CH2-
CO-NH-trityl), 1.72 (s, 3H, thymine-CH3). 13C NMR (125 MHz,
DMSO): δ 172.2, 169.1, 168.0, 165.1, 157.0, 151.7, 145.6, 145.5,
144.5, 143.0, 141.4, 129.2, 128.3, 128.1, 127.8, 127.0, 125.8, 120.8,
108.7, 69.9, 69.0, 67.7, 66.2, 47.4, 28.1, 25.8, 12.6. LRMS (ESI-MS
m/z) mass calcd for C48H45N5O8 [M - H]-, 818.32; found, 818.3.
(S)-2-(N-(2-(((4H-Fluoren-9-yl)methoxy)carbonylamino)ethyl)-2-
(thymin-1( 2H)-yl)acetamido)-6-(tert-butoxycarbonylamino)hexano-
ic Acid (9a). Following the procedure used to prepare compound 5a,
8a (426 mg) was converted to 347 mg (85%) of 9a as a flaky off-
1
white solid. H NMR (500 MHz, DMSO): δ 11.33 (s, 1H, imide-
NH), 7.86 (d, J ) 10 Hz, 2H, Fmoc-H), 7.67 (d, J ) 10 Hz, 2H,
Fmoc-H), 7.28-7.42 (m, 5H, Fmoc-H + thymine-H), 6.77 (m, 1H,
Fmoc-NH), 4.65 (s, 2H, thymine-CH2-CO), 4.35 (d, J ) 5 Hz, 2H,
Fmoc-CH-CH2-O), 4.29 (m, 1H, N-CH-CO2H), 4.22 (t, J ) 2.5
Hz, 1H, Fmoc-CH-CH2), 3.37 (m, 2H, Fmoc-NH-CH2-CH2-NH),
3.25 (m, 2H, Fmoc-NH-CH2-CH2), 2.88 (m, 2H, Boc-NH-CH2-
CH2) 1.74 (s, 3H, thymine-CH3), 1.10-1.50 (m, 6H, CH-CH2-CH2-
CH2-CH2), 1.34 (s, 9H, tert-butyl-CH3). 13C NMR (125 MHz,
DMSO): δ 171.3, 165.2, 156.4, 151.9, 144.7, 141.6, 132.9, 132.1,
129.7, 128.4, 128.0, 125.9, 120.8, 108.8, 78.1, 66.2, 60.3, 55.8, 47.7,
29.0, 21.6, 15.0, 12.6. HRMS (ESI-MS m/z): mass calcd for C38H47N5O9
[M - H]-, 676.30; found, 676.30.
(R)-2-(N-(2-(((9H-Fluoren-9-yl)methoxy)carbonylamino)ethyl)-2-
(thymin-1(2H)-yl)acetamido)-5-oxo-5-(tritylamino)pentanoic Acid.
All characterization data matched 9d.
(R)-2-(N-(2-(((9H-Fluoren-9-yl)methoxy)carbonylamino)ethyl)-2-
(thymin-1(2H)-yl)acetamido)-5-tert-butoxy-5-oxopentanoic Acid (9e).
1H NMR (500 MHz, DMSO): δ 11.29 (s, 1H, imide-NH), 7.87 (m,
2H, Fmoc-H), 7.66 (d, J ) 10 Hz, 2H, Fmoc-H), 7.28-7.42 (m,
5H, Fmoc-H + thymine-H), 6.86 (s, 1H, Fmoc-NH), 4.63 (m, 2H,
thymine-CH2-CO), 4.33 (d, 2H, J ) 10 Hz, Fmoc-CH-CH2-O),
4.18-4.29 (m, 2H, N-CH-CO2H + Fmoc-CH-CH2), 3.44 (m, 2H,
Fmoc-NH-CH2-CH2-N), 3.25 (m, 2H, Fmoc-NH-CH2-CH2),
2.22 (t, 2H, J ) 10 Hz, CH2-CH2-CO2t-Bu), 2.08-2.18 (m, 2H, CH-
CH2-CH2-CO2t-Bu), 1.73 (s, 3H, thymine-CH3), 1.36 (m, 9H, tert-
butyl-CH3). 13C NMR (125 MHz, DMSO): δ 172.5, 168.0, 165.1,
157.0, 151.7, 144.5, 143.0, 141.4, 128.3, 127.7, 125.8, 120.8, 119.1,
108.6, 80.4, 66.1, 59.8, 48.6, 47.5, 32.2, 30.9, 28.1, 24.8, 12.6. LRMS
(ESI-MS m/z) mass calcd for C33H38N4O9 [M - H]-, 633.25; found,
633.2.
The following R-functionalized thymine monomers were prepared
analogously.
(R)-2-(N-(2-(((9H-Fluoren-9-yl)methoxy)carbonylamino)ethyl)-2-
(thymin-1(2H)-yl)acetamido)-6-(tert-butoxycarbonylamino)hexano-
ic Acid. All characterization data matched 9a.
(R)-2-(N-(2-(((9H-Fluoren-9-yl)methoxy)carbonylamino)ethyl)-2-
(thymin-1(2H)-yl)acetamido)-3-phenylpropanoic Acid (9b). 1H NMR
(500 MHz, DMSO): δ 11.32 (s, 1H, imide-NH), 7.86 (m, 2H, Fmoc-
H), 7.62 (d, J ) 10 Hz, 2H, Fmoc-H), 7.08-7.42 (m, 10H, Fmoc-H
9
J. AM. CHEM. SOC. VOL. 130, NO. 14, 2008 4657

A R T I C L E S
Kleiner et al.
(S)-2-(N-(2-(((9H-Fluoren-9-yl)methoxy)carbonylamino)ethyl)-2-
(thymin-1(2H)-yl)acetamido)-5-tert-butoxy-5-oxopentanoic Acid. All
characterization data matched 9e.
evaporated under reduced pressure to yield 352 mg (80.0%) of 13a as
a yellow solid. H NMR (500 MHz, DMSO): δ major rotamer 11.33
1
(s, 1H, imide-NH), 7.87 (d, J ) 5 Hz, 2H, Fmoc-H), 7.69 (d, J ) 5
Hz, 2H, Fmoc-H), 7.35 (m, 4H, Fmoc-H), 7.17 (s, 1H, thymine-
H), 6.75 (m, 1H, Fmoc-NH), 4.65 (m, 2H, thymine-CH2-CO), 4.43
(m, 2H, N-CH2-CO2H), 4.33 (m, 1H, Fmoc-NH-CH-CH2), 4.25
(d, J ) 5 Hz, 2H, Fmoc-CH-CH2-O), 4.20 (t, J ) 5 Hz, 1H, Fmoc-
CH-CH2), 3.35 (m, 2H, Fmoc-NH-CH-CH2-N), 2.86 (m, 2H,
CH2-CH2-NH-Boc), 1.69 (s, 3H, thymine-CH3), 1.10-1.50 (m, 6H,
CH-CH2-CH2-CH2-CH2), 1.35 (s, 9H, tert-butyl-CH3); minor
rotamer 11.35 (s, 1H, imide-NH), 7.20 (s, 1H, thymine-H), 1.67 (s,
3H, thymine-CH3). 13C NMR (125 MHz, DMSO): δ 174.0, 165.1,
156.3, 151.7, 140.1, 136.1, 130.2, 129.6, 128.0, 110.5, 84.0, 65.5, 49.2,
45.6, 31.6, 30.8, 29.7, 29.2, 28.9, 28.1, 26.9, 22.7, 18.1, 12.6. LRMS
(ESI-MS m/z) mass calcd for C38H47N5O9 [M - H]-, 676.30; found,
676.2.
Synthesis of γ-Functionalized Thymine Fmoc-Peptide Nucleic
Acid (PNA) Monomers. The synthesis of γ-functionalized Fmoc-PNA
thymine monomers was adapted from the previously described synthesis
of the Boc-γ-L-lysine(Fmoc) thymine monomer.24 The representative
synthesis of the γ-D-lysine-thymine monomer is presented below.
tert-Butyl (R)-5-(((9H-Fluoren-9-yl)methoxy)carbonylamino)-6-
oxohexyl-carbamate (10a). This compound was prepared according
to the protocols of Wang et al.46
(R)-Benzyl 2-(2-(((4H-Fluoren-9-yl)methoxy)carbonylamino)-6-
(tert-butoxycarbonylamino)hexylamino)acetate (11a). To a stirring
solution of 10a (965 mg) and benzyl glycinate hydrochloride (473 mg)
in 15 mL of CH2Cl2 at RT was added N,N-diisopropylethylamine (410
µL) over 2 min. After stirring for 10 min, sodium triacetoxyborohydride
(630 mg) was added. After stirring for 90 min, 15 mL of saturated
aqueous Na2CO3 was added to quench the reaction. After stirring for
10 min, the reaction mixture was extracted with CH2Cl2. The combined
organic layers were dried over MgSO4 and evaporated under reduced
pressure. The resulting oil was purified by flash column chromatography
[Rf ) 0.50 (EtOAc)] to yield 662 mg (51.7%) of 11a as a clear, colorless
The following γ-functionalized thymine monomers were prepared
analogously.
(S)-2-(N-(2-(((4H-Fluoren-9-yl)methoxy)carbonylamino)-6-(tert-
butoxycarbonylamino)hexyl)-2-(thymin-1(2H)-yl)acetamido)ace-
tic Acid. All characterization data matched 13a.
(R)-2-(N-(2-(((9H-Fluoren-9-yl)methoxy)carbonylamino)-3-phe-
nylpropyl)-2-(thymin-1(2H)-yl)acetamido)acetic Acid (13b). 1H NMR
(500 MHz, DMSO): δ major rotamer 11.24 (s, 1H, imide-NH), 7.85
(m, 2H, Fmoc-H), 7.50-7.62 (m, 2H, Fmoc-H), 7.10-7.42 (m, 10H,
Fmoc-H + thymine-H + phenyl-H), 4.68 (m, 2H, thymine-CH2-
CO), 3.96-4.80 (m, 6H, N-CH2-CO2H + Fmoc-NH-CH-CH2 +
Fmoc-CH-CH2-O + Fmoc-CH-CH2), 3.30-3.80 (m, 2H, Fmoc-
NH-CH-CH2-N), 2.64-3.06 (m, 2H, phenyl-CH2-CH), 1.70 (m,
3H, thymine-CH3); minor rotamer 11.31 (s, 1H, imide-NH). 13C NMR
(125 MHz, DMSO): δ 165.1, 163.0, 151.7, 149.3, 142.8, 141.4, 140.6,
139.3, 139.0, 136.1, 130.2, 130.1, 129.8, 129.6, 129.3, 129.1, 128.9,
128.8, 128.7, 128.4, 128.3, 128.0, 127.7, 125.9, 124.9, 124.6, 122.1,
121.9, 120.8, 120.6, 108.8, 78.9, 47.3, 42.5, 36.5, 31.5, 31.4, 28.1, 18.8,
12.6. LRMS (ESI-MS m/z) mass calcd for C33H32N4O7 [M - H]-,
595.22; found, 595.2.
(S)-2-(N-(2-(((9H-Fluoren-9-yl)methoxy)carbonylamino)-3-phe-
nylpropyl)-2-(thymin-1(2H)-yl)acetamido)acetic Acid. All charac-
terization data matched 13b.
(S)-2-(N-(2-(((9H-Fluoren-9-yl)methoxy)carbonylamino)-4-meth-
ylpentyl)-2-(thymin-1(2H)-yl)acetamido)acetic Acid (13c). 1H NMR
(500 MHz, DMSO): δ major rotamer 11.29 (s, 1H, imide-NH), 7.87
(m, 2H, Fmoc-H), 7.68 (m, 2H, Fmoc-H) 7.1-7.4 (m, 5H, Fmoc-H
+ thymine-H), 6.26 (s, 1H, Fmoc-NH), 4.67 (m, 2H, thymine-CH2-
1
oil. H NMR (500 MHz, CDCl3): δ 7.71 (d, J ) 10 Hz, 2H, Fmoc-
H), 7.58 (m, 2H, Fmoc-H), 7.25-7.37 (m, 9H, Fmoc-H + phenyl-
H), 5.24 (br d, 1H, J ) 10 Hz, Fmoc-NH), 5.12 (s, 2H, O-CH2-
phenyl), 4.80 (br s, 1H, Boc-NH), 4.39 (d, J ) 5 Hz, 2H, Fmoc-
CH-CH2-O), 4.18 (t, J ) 5 Hz, 1H, Fmoc-CH-CH2), 3.65 (s, 1H,
Fmoc-NH-CH-CH2), 3.41 (m, 2H, NH-CH2-CO2Bn), 3.06 (d, J
) 10 Hz, 2H, Boc-NH-CH2-CH2), 2.61 (m, 2H, Fmoc-NH-CH-
CH2-NH), 1.2-1.5 (m, 6H, CH-CH2-CH2-CH2-CH2), 1.43 (s, 9H,
tert-butyl-CH3). 13C NMR (125 MHz, CDCl3): δ 172.4, 156.8, 156.3,
144.1, 141.8, 135.9, 128.7, 128.5, 128.1, 127.7, 125.2, 79.4, 66.8, 66.3,
53.0, 51.4, 47.9, 47.0, 40.3, 33.0, 30.4, 28.8, 23.3. HRMS (ESI-MS
m/z) mass calcd for C35H43N3O6 [M + H]+, 602.32; found, 602.32.
(R)-Benzyl 2-(N-(2-(((4H-Fluoren-9-yl)methoxy)carbonylamino)-
6-(tert-butoxycarbonylamino)hexyl)-2-(thymin-1(2H)-yl)acetamido)-
acetate (12a). To a stirring solution of 11a (662 mg), 1-thymine acetic
acid (262 mg), and HOBt (8.13 mg) in 23 mL of dry DMF at RT was
added EDC (315 mg). After stirring overnight, H2O was added, forming
a white precipitate. The reaction mixture was extracted into EtOAc.
The combined organic layers were subsequently washed with 1 M
aqueous HCl, saturated aqueous NaHCO3, H2O, and brine. The organic
layer was dried over MgSO4 and evaporated under reduced pressure
to yield 500 mg (59.2%) of 12a as a white solid. 1H NMR (500 MHz,
DMSO): δ major rotamer 11.33 (s, 1H, imide-NH), 7.86 (m, 2H,
Fmoc-CH), 7.67 (m, 2H, Fmoc-CH), 7.35 (m, 9H, phenyl-H +
Fmoc-H), 7.19 (s, 1H, thymine-H), 6.79 (m, 1H, Fmoc-NH), 5.20
(d, 2.5 Hz, 1H, Boc-NH), 5.12 (s, 2H, O-CH2-phenyl), 4.73 (m,
2H, thymine-CH2-CO), 4.26-4.40 (m, 3H, Fmoc-NH-CH-CH2
+ Fmoc-CH-CH2-O), 4.21 (t, J ) 10 Hz, 1H, Fmoc-CH-CH2),
4.11 (m, 2H, N-CH2-CO2Bn), 3.33 (m, 2H, Fmoc-NH-CH-CH2-
N), 2.91 (m, 2H, CH2-CH2-NH-Boc), 1.69 (s, 3H, thymine-CH3),
1.10-1.50 (m, 6H, CH-CH2-CH2-CH2-CH2), 1.36 (s, 9H, tert-
butyl-CH3), minor rotamer 11.32 (s, 1H, imide-NH), 1.72 (s, 3H,
thymine-CH3). 13C NMR (125 MHz, DMSO): δ 169.8, 168.6, 165.0,
156.1, 156.7, 152.0, 144.2, 142.3, 141.8, 128.8, 128.7, 128.3, 128.0,
125.8, 120.6, 109.0, 77.9, 66.8, 66.5, 66.3, 60.4, 52.1, 50.2, 47.8, 29.9,
28.8, 23.7, 21.6, 15.0, 12.6. HRMS (ESI-MS m/z) mass calcd for
C42H49N5O9 [M + H]+, 768.36; found, 768.36.
CO), 3.8-4.5 (m, 6H, N-CH2-CO2H + Fmoc-NH-CH-CH2
+
Fmoc-CH-CH2-O + Fmoc-CH-CH2), 3.30-3.52 (m, 2H, Fmoc-
NH-CH-CH2-N), 1.70 (s, 1H, thymine-CH3), 1.2-1.6 (m, 3H,
CH3-CH-CH3 + CH-CH2-CH), 0.84 (t, J ) 20 Hz, 6H, CH3-
CH-CH3), minor rotamer 11.27 (s, 1H, imide-NH), 1.72 (s, 1H,
thymine-CH3), 0.82 (t, J ) 20 Hz, 6H, CH3-CH-CH3). 13C NMR
(125 MHz, DMSO): δ 171.4, 171.0, 165.1, 156.7, 156.6, 151.7, 144.8,
144.6, 144.5, 144.4, 142.6, 141.5, 128.3, 127.7, 125.9, 125.8, 124.9,
120.8, 120.7, 110.9, 108.8, 65.9, 65.8, 52.4, 52.1, 48.6, 48.4, 48.3, 47.6,
31.1, 25.0, 24.1, 22.4, 22.1, 12.6. LRMS (ESI-MS m/z) mass calcd for
C30H34N4O7 [M - H]-, 561.23; found, 561.2.
(R)-2-(N-(2-(((9H-Fluoren-9-yl)methoxy)carbonylamino)-4-meth-
ylpentyl)-2-(thymin-1(2H)-yl)acetamido)acetic Acid. All characteriza-
tion data matched 13c.
(R)-2-(N-(2-(((9H-Fluoren-9-yl)methoxy)carbonylamino)-5-oxo-
5-(tritylamino)pentyl)-2-(thymin-1(2H)-yl)acetamido)acetic Acid (13d).
1H NMR (500 MHz, DMSO): δ major rotamer 11.28 (s, 1H, imide-
NH), 8.59 (s, 1H, trityl-NH), 7.88 (d, J ) 9.0 Hz, 2H, Fmoc-H),
7.68 (m, 2H, Fmoc-H), 7.10-7.42 (m, 20H, thymine-H + trityl-H
+ Fmoc-H), 6.85 (s, 1H, Fmoc-NH), 4.64 (m, 2H, thymine-CH2-
CO), 4.16-4.36 (m, 3H, Fmoc-CH-CH2-O + Fmoc-CH-CH2),
3.92 (m, 2H, N-CH2-CO2H), 3.24-3.44 (m, 3H, Fmoc-NH-CH-
(R)-2-(N-(2-(((4H-Fluoren-9-yl)methoxy)carbonylamino)-6-(tert-
butoxycarbonylamino)hexyl)-2-(thymin-1(2H)-yl)acetamido)ace-
tic Acid (13a). To a stirring solution of 12a (748 mg) in 91.3 mL of
dry methanol was added Pd/C (183 mg) over 2 min. The reaction
mixture was placed under H2 (balloon pressure). After stirring overnight,
TLC analysis showed the disappearance of starting material [Rf ) 0.40
(EtOAc)], and the reaction mixture was filtered through celite and
9
4658 J. AM. CHEM. SOC. VOL. 130, NO. 14, 2008

DNA-Templated Polymerization
A R T I C L E S
CH2 + Fmoc-NH-CH-CH2-N), 2.16-2.42 (4H, CH-CH2-CH2-
CO-NH-trityl), 1.64 (s, 1H, thymine-CH3); minor rotamer 11.25 (s,
1H, imide-NH), 8.53 (s, 1H, trityl-NH), 1.66 (s, 1H, thymine-CH3).
13C NMR (125 MHz, DMSO): δ 172.2, 170.9, 168.1, 156.9, 151.6,
145.6, 144.6, 144.5, 142.5, 141.4, 129.2, 128.3, 128.1, 127.8, 127.0,
125.9, 125.6, 120.8, 108.9, 69.9, 66.1, 47.5, 33.2, 31.1, 12.6. LRMS
(ESI-MS m/z) mass calcd for C48H45N5O8 [M - H]-, 818.32; found,
818.3.
polymerization: 5′-H2N-CGCCTTCGTCCTCGAAGGCGAACCCA-
CCCACCCACCCACCCCACACACACACACACACACA-3′. Reac-
tions were heated to 95 °C and then slowly cooled to the reaction
temperature. Reductive amination was initiated by the addition of 1
µL of 4 M aqueous sodium cyanoborohydride, and reactions were
quenched at the indicated time by addition of 2.5 µL of neat (13.3 M)
allyl amine. The quenched reactions were desalted using Princeton
Separation sephadex minicolumns and separated by denaturing PAGE
on a 15% TBE/urea polyacrylamide gel. Denaturing polyacrylamide
gels were stained with ethidium bromide and visualized by UV
illumination and densitometry using standard procedures.
(S)-2-(N-(2-(((9H-Fluoren-9-yl)methoxy)carbonylamino)-5-oxo-
5-(tritylamino)pentyl)-2-(thymin-1(2H)-yl)acetamido)acetic Acid. All
characterization data matched 13d.
(R)-2-(N-(2-(((9H-Fluoren-9-yl)methoxy)carbonylamino)-5-tert-
butoxy-5-oxopentyl)-2-(thymin-1(2H)-yl)acetamido)acetic Acid (13e).
1H NMR (500 MHz, DMSO): δ major rotamer 11.29 (s, 1H, imide-
NH), 7.88 (d, J ) 10 Hz, 2H, Fmoc-H), 7.68 (m, 2H, Fmoc-H),
7.12-7.43 (m, 5H, Fmoc-H + thymine-H), 6.86 (s, 1H, Fmoc-NH),
4.66 (m, 2H, thymine-CH2-CO), 4.1-4.5 (m, 6H, N-CH2-CO2H
+ Fmoc-NH-CH-CH2 + Fmoc-CH-CH2-O + Fmoc-CH-CH2),
3.26-3.60 (m, 2H, Fmoc-NH-CH-CH2-N), 2.16 (m, 2H, CH2-
CH2-CO2t-Bu), 1.74 (m, 2H, CH2-CH2-CH), 1.68 (s, 3H, thymine-
CH3), 1.36 (m, 9H, tert-butyl-CH3); minor rotamer 11.27 (s, 1H,
imide-NH), 1.71 (s, 3H, thymine-CH3). 13C NMR (125 MHz,
DMSO): δ 172.6, 170.1, 168.1, 165.1, 156.8, 151.6, 144.4, 142.5,
141,1, 139.9, 128.3, 127.7, 125.9, 125.6, 120.8, 108.9, 80.3, 67.7, 66.0,
48.4, 47.5, 35.1, 32.2, 31.1, 28.4, 25.8, 21.7, 12.6. LRMS (ESI-MS
m/z) mass calcd for C33H38N4O9 [M - H]-, 633.25; found, 633.2.
(S)-2-(N-(2-(((9H-Fluoren-9-yl)methoxy)carbonylamino)-5-tert-
butoxy-5-oxopentyl)-2-(thymin-1(2H)-yl)acetamido)acetic Acid. All
characterization data matched 13e.
Synthesis of PNA Pentamer Aldehydes. PNA pentamer aldehydes
were synthesized using preloaded H-Thr-Gly-NovaSyn TG resin
(Novabiochem) and standard automated Fmoc solid-phase peptide
synthesis on an Applied Biosystems 433A peptide synthesizer by
adapting previously described protocols.18 PNA pentamer aldehydes
were purified by reverse-phase high-pressure liquid chromatography
(HPLC) using a C18 stationary phase and an acetonitrile/0.1%
trifluoroacetic acid (TFA) gradient, characterized by electrospray mass
spectrometry on a Waters Q-TOF Premier instrument, and quantitated
using UV spectroscopy carried out on a Nanodrop ND1000 spectro-
photometer.
DNA-Templated Reductive Amination Protocol for Tetramer
Building Blocks. Polymerization reactions were prepared by mixing
1 µL of 500 mM TAPS pH 8.5 buffer, 1.25 µL of 4 M NaCl, 20 pmol
of DNA template, and 800 pmol of PNA tetramer aldehyde with water
to a total volume of 50 µL. DNA template sequence for H2N-ggtt-
CHO and H2N-ggt*t-CHO matched codon polymerization: 5′-H2N-TCG-
CCTTCGTCCTCGAAGGCGAAACCAACCAACCAACCAACCAA-
CCAACCAACCAACCAACC-3′. DNA template sequence for H2N-
ggtt-CHO and H2N-ggt*t-CHO mismatched codon polymerization: 5′-
H2N- TCGCCTTCGTCCTCGAAGGCGAAACCAACCAACCAACC-
AACCACACACACACACACACACAC-3′. DNA template sequence
for H2N-gggt-CHO and H2N-ggg*t-CHO matched codon polymeriza-
tion: 5′-H2N-TCGCCTTCGTCCTCGAAGGCGAACCCACCCACCC-
ACCCACCCACCCACCCACCCACCCACCC-3′. DNA template se-
quence for H2N-gggt-CHO and H2N-ggg*t-CHO mismatched codon
DNA-Templated Reductive Amination Protocol for Pentamer
Building Blocks. Polymerization reactions were prepared by mixing
1 µL of 500 mM TAPS pH 8.5 buffer, 1.25 µL of 4 M NaCl, 20 pmol
of DNA template, and 640 pmol of PNA pentamer aldehyde with water
to a total volume of 50 µL. DNA template sequence for matched codon
polymerization: 5′-H2N-TCGCCTTCGTCCTCGAAGGCGAAATC-
CAATCCAATCCAATCCAATCCAATCCAATCCAATCC-3′. DNA
template sequence for mismatched codon polymerization: 5′-H2N-TCG-
CCTTCGTCCTCGAAGGCGAAATCCAATCCAATCCAATCCAT-
ACCATACCATACCATACC-3′. Reactions were heated to 95 °C and
then slowly cooled to the reaction temperature. Reductive amination
was initiated by addition of 1 µL of 4 M aqueous sodium cyanoboro-
hydride, and reactions were quenched at the indicated time by addition
of 2.5 µL of neat (13.3 M) allyl amine. The quenched reactions were
desalted using Princeton Separation sephadex minicolumns and sepa-
rated by denaturing PAGE on a 15% TBE/urea polyacrylamide gel.
Denaturing polyacrylamide gels were stained with ethidium bromide
and visualized by UV illumination and densitometry using standard
procedures.
Acknowledgment. Dedicated to the memory of Leslie E.
Orgel (1927-2007), whose research and ideas on chemical
evolution were inspirational to those who continue to work in
this area. This research was supported by the Office of Naval
Research (N00014-03-1-0749), NIH Grant R01GM065865, and
the Howard Hughes Medical Institute. We thank Dr. Yinghua
Shen and Walter E. Kowtoniuk for assistance with mass
spectrometry and Leslie Vogt for assistance with molecular
modeling. Y.B. gratefully acknowledges the support of an NSF
Graduate Research Fellowship. M.B. thanks the Harvard College
Research Program and the Pechet Family Fund for Undergradu-
ate Research for support.
Supporting Information Available: ESI-MS characterization
of PNA tetramer and pentamer aldehydes, MALDI-TOF char-
acterization of DNA-templated polymer products, polymeriza-
tion of functionalized tetramer aldehydes, molecular modeling
protocols, data for melting experiments, and 1H and 13C spectra
for all compounds listed in the Experimental Section. This
material is available free of charge via the Internet at
http://pubs.acs.org.
JA0753997
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