Synthesis and nucleic acids binding properties of diastereomeric aminoethylprolyl peptide nucleic acids (aepPNA)

Article abstract of DOI:10.1080/15257770.2010.547839

A general synthetic method for Fmoc-protected monomers of all four diastereomeric aminoethyl peptide nucleic acid (aepPNA) has been developed. The key reaction is the coupling of nucleobase-modified proline derivatives and Fmoc-protected aminoacetaldehyde by reductive alkylation. Oligomerization of the aepPNAs up to 10mer was achieved by Fmoc-solid phase peptide synthesis methodology. Preliminary binding studies of these aepPNA oligomers with nucleic acids suggested that the "cis-" homothymine aepPNA decamers with (2′R,4′R) and (2′S,4′S) configurations can bind, albeit with slow kinetics, to their complementary RNA [poly(adenylic acid)] but not to the complementary DNA [poly(deoxyadenylic acid)]. On the other hand, the trans homothymine aepPNA decamers with (2′R,4′S) and (2′S,4′R) configurations failed to form stable hybrid with poly(adenylic acid) and poly(deoxyadenylic acid). No hybrid formation could be observed between a mixed-base (2′R,4′R)-aepPNA decamer with DNA and RNA in both antiparallel and parallel orientations. Copyright Taylor and Francis Group, LLC.

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Synthesis and Nucleic Acids Binding
Properties of Diastereomeric
Aminoethylprolyl Peptide Nucleic Acids
(aepPNA)
Patcharee Ngamwiriyawong a & Tirayut Vilaivan a
a Organic Synthesis Research Unit, Department of Chemistry, Faculty
of Science , Chulalongkorn University , Bangkok , Thailand
Published online: 24 Feb 2011.
To cite this article: Patcharee Ngamwiriyawong & Tirayut Vilaivan (2011) Synthesis and Nucleic Acids
Binding Properties of Diastereomeric Aminoethylprolyl Peptide Nucleic Acids (aepPNA), Nucleosides,
Nucleotides and Nucleic Acids, 30:2, 97-112, DOI: 10.1080/15257770.2010.547839
To link to this article: http://dx.doi.org/10.1080/15257770.2010.547839
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Nucleosides, Nucleotides and Nucleic Acids, 30:97–112, 2011
Copyright ꢀC Taylor and Francis Group, LLC
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770.2010.547839
SYNTHESIS AND NUCLEIC ACIDS BINDING PROPERTIES
OF DIASTEREOMERIC AMINOETHYLPROLYL PEPTIDE NUCLEIC
ACIDS (aepPNA)
Patcharee Ngamwiriyawong and Tirayut Vilaivan
Organic Synthesis Research Unit, Department of Chemistry, Faculty of Science,
Chulalongkorn University, Bangkok, Thailand
A general synthetic method for Fmoc-protected monomers of all four diastereomeric aminoethyl
2
peptide nucleic acid (aepPNA) has been developed. The key reaction is the coupling of nucleobase-
modified proline derivatives and Fmoc-protected aminoacetaldehyde by reductive alkylation.
Oligomerization of the aepPNAs up to 10mer was achieved by Fmoc-solid phase peptide synthesis
methodology. Preliminary binding studies of these aepPNA oligomers with nucleic acids suggested
that the “cis-” homothymine aepPNA decamers with (2ꢁR,4ꢁR) and (2ꢁS,4ꢁS) configurations can
bind, albeit with slow kinetics, to their complementary RNA [poly(adenylic acid)] but not to the com-
plementary DNA [poly(deoxyadenylic acid)]. On the other hand, the trans homothymine aepPNA
decamers with (2ꢁR,4ꢁS) and (2ꢁS,4ꢁR) configurations failed to form stable hybrid with poly(adenylic
acid) and poly(deoxyadenylic acid). No hybrid formation could be observed between a mixed-base
(2ꢁR,4ꢁR)-aepPNA decamer with DNA and RNA in both antiparallel and parallel orientations.
Keywords PNA; RNA; hybridization; triplex forming oligonucleotides
INTRODUCTION
Peptide nucleic acid is a structural mimic of DNA that exhibits interest-
ing base-pairing properties.[1] Since the first introduction of aminoethylgly-
cyl (aeg) PNA in 1991,[2] a number of modifications of the original aegPNA
backbone have been proposed with the aim to improve certain properties
such as binding affinity, solubility and cell penetration. A considerable suc-
cess has been made by restriction of the original aegPNA conformations
by incorporating one or more ring structures in the backbone.[3,4] Some
of these conformationally restricted PNA exhibited comparable or even
Received 12 October 2010; accepted 10 December 2010.
Financial support for this work comes from the Thailand Research Fund (RTA5280002) and Chu-
lalongkorn University (Rachadapisek Sompoj Endowment to OSRU).
Address correepondence to Tirayut Vilaivan, Organic Synthesis Research Unit, Department of
Chemistry, Faculty of Science, Chulalongkorn University, Phayathai Road, Patumwan, Bangkok 10330,
Thailand. E-mail: vtirayut@chula.ac.th
97

98
P. Ngamwiriyawong and T. Vilaivan
B
B
O
O
O
N
N
N
N
H
H
n
n
aepPNA
aegPNA
FIGURE 1 Structures of aegPNA and aepPNA (stereochemistry not shown).
stronger affinity to DNA and/or RNA, while the high specificity of the base-
pairing was still retained. Furthermore, few modified PNA even possess extra
features not present in the original Nielsen’s aegPNA such as preference for
binding to DNA in one specific direction and preferential binding to specific
types of nucleic acids (DNA or RNA)[5–7]
.
Since 1999, at least three research groups have independently reported
syntheses and hybridization studies of a new conformationally constrained
and positively charged PNA known as aminoethylprolyl PNA (aepPNA,
Figure 1). The presence of a positive charge on the non-acylated pyrrolidine
nitrogen atom (pKa ∼ 6.5)[8] in the PNA backbone could potentially im-
prove the solubility and binding affinity to DNA, which is negatively charged.
D’Costa and coworkers have studied two of the four possible stereoisomers
of aepPNA. They reported that the homothymine aepPNA octamer with
(2ꢁS,4ꢁS) and (2ꢁR,4ꢁS) stereochemistry formed very stable (PNA)2·DNA
triplexes with their complementary DNA (Tm > 80◦C).[8] They also re-
ported a site-specific incorporation of aepPNA monomers into Nielsen’s
aegPNA oligomers by solid phase synthesis. These chimeric aeg-aepPNAs
were shown to have improved antiparallel vs parallel directional selectivity
toward DNA compared to the unmodified aegPNA, while the binding affin-
ity and specificity was retained.[9,10] Liu and coworkers reported the synthe-
sis of nonchimeric aepPNA oligomers with (2ꢁS,4ꢁS) stereochemistry.[11] In
contrast to earlier findings,[8] The non-chimeric homothymine and mixed-
sequence aepPNA showed no detectable binding with complementary DNA
according to UV melting and titration.[11] Our study on aepPNA oligomer
with a different stereochemistry, namely (2ꢁR,4ꢁR), revealed that the homoth-
ymine aepPNA decamer forms a fairly stable hybrid with complementary
RNA, whereas no binding to the complementary DNA could be observed.[12]
The hybridization properties of aepPNA oligomers reported so far
showed some discrepancies, which may be sequence and stereochemistry
dependent. None of these research groups has yet synthesized all four pos-
sible stereoisomers and perform a systematic comparison under identical
conditions. In addition, work from other groups involving aepPNA mostly
emphasized on its DNA binding properties. In view of our previous success
in developing a PNA that binds preferentially to DNA,[5,6] it would be de-
sirable to have an alternative PNA system that binds favorably, or at least

Synthesis and Nucleic Acids Binding Properties of aepPNA
99
equally well, to RNA. These RNA binding PNAs would have a great potential
in antisense applications.[1] Toward this goal, a general synthetic route to-
ward the PNA momomers and oligomers first needs to be developed. In this
paper, we report a successful synthesis of all four stereoiosomers of Fmoc-
protected aepPNA monomers employing a new reductive alkylation strategy.
We also report a synthesis of the aepPNA up to 10mer from these monomers
as well as a preliminary binding study between these diastereomeric aepPNA
oligomers and DNA/RNA.
RESULTS AND DISCUSSION
Synthesis of Monomers
Ganesh[8,9] and Lui[11] had previously developed a method for
the synthesis of Boc-protected aepPNA monomers. N -alkylation of 4-
hydroxyproline esters with N -Boc-bromoethylamine afforded N -(N -Boc-
aminoethyl)hydroxyproline intermediates in 60–68% yield. These hydroxyl
compounds were activated by mesylation and then displaced with an ap-
propriately protected nucleobase to give the N -Boc protected aepPNA
monomers in moderate yields. In our previous report, a different approach
had been taken whereby a nucleobase-modified proline was N -alkylated
with N -nosylaziridine.[12] Exchanging the N -protecting groups from nosyl to
Fmoc via the Boc-protected intermediate provided the expected monomers.
These methods, however, suffer from providing low yield and require many
steps.
Reductive alkylation of amino acids by N -protected aminoaldehydes in
the presence of NaBH3CN had been widely used in the synthesis of N -
aminoethylated amino acids, including aegPNA monomers.[13–16] We pro-
posed that the reductive alkylation of nucleobase-substituted proline with
N -Fmoc-aminoacetaldehyde should directly provide the Fmoc-protected
aepPNA monomers without the need for protecting group conversion
(Scheme 1).
N -Fmoc-aminoacetaldehyde (1) was easily prepared in two steps with
90% overall yield from NaIO4 oxidation of 3-amino-1,2-propanediol.[16,17]
The Boc group in the N -Boc thymine-modified proline derivative (2a) was
selectively removed in the presence of the diphenylmethyl ester by treatment
with 3 equivalents of p-toluenesulfonic acid (p-TsOH) in acetonitrile at am-
bient temperature.[18] The reductive alkylation of the free amine with 1 took
place in the presence of NaBH3CN as a reductant and NaOAc as a buffer-
ing agent. The desired product (3a) was obtained as a white foam in 76%
overall yield (two steps from 2a and 1). Spectroscopic data of the product
were identical to those of the same compound obtained via alkylation with
aziridine followed by protecting group conversion.[12] Other Boc-protected

100
P. Ngamwiriyawong and T. Vilaivan
B1
B1
B1
4'
O
O
Dpm
O
i
ii
DpmO
N
N
H
DpmO
N
2'
H
OTs
Boc
NHFmoc
3a-3g
2a-2g
(ref 18)
B2
O
i
pTsOH, MeCN
iii
HO
ii FmocNHCH2CHO, NaBH3CN, NaOAc
iii TFA, anisole
N
NHFmoc
4a-4g
SCHEME 1 Synthesis of aepPNA monomers via reductive alkylation.
nucleobase-modified proline derivatives (2b–2g)[18] were then subjected to
the same reductive alkylation with 1 under similar conditions. In all cases
the expected products 3b–3g were obtained in moderate to good yields
(Table 1). Although no satisfactory explanation could be offered, it is
interesting to note that the reductive alkylation of the “trans” (2ꢁR,4ꢁS)
and (2ꢁS,4ꢁR) isomers consistently gave poorer yield compared to the “cis”
(2ꢁR,4ꢁR) and (2ꢁS,4ꢁS) isomers (Table 1, compare entries 3, 4, and 1, 2).
To obtain the Fmoc-protected free acids 4, the diphenylmethyl ester
group in 3a–3g was next removed by treatment with trifluoroacetic acid
in the presence of anisole at ambient temperature. The free acids 4a–4g
were obtained in 72–85% yield (Table 1). In case of the thymine monomers
4a–4d, the N 3-benzoyl group on the thymine was simultaneously cleaved
under these conditions. As protection of the thymine-N 3 was not generally
required during the PNA synthesis, this premature cleavage would not be
considered as a serious problem.
TABLE 1 Synthesis of protected PNA monomers by reductive alkylation with N -Fmoc-
aminoacetaldehyde
Starting
material
Entry
Config.
Product
B1
Yield (%)
Product
B2
Yield (%)
1
2
3
4
5
6
7
2a
2b
2c
2d
2e
2f
(2ꢁR,4ꢁR)
(2ꢁS,4ꢁS)
(2ꢁR,4ꢁS)
(2ꢁS,4ꢁR)
(2ꢁR,4ꢁR)
(2ꢁR,4ꢁR)
(2ꢁR,4ꢁR)
3a
3b
3c
3d
3e
3f
TBz
TBz
TBz
TBz
GIbu
ABz
CBz
76
87
40
43
69
60
89
4a
4b
4c
4d
4e
4f
T
T
T
85
72
72
75
77
75
74
T
GIbu
ABz
CBz
2g
3g
4g

Synthesis and Nucleic Acids Binding Properties of aepPNA
101
Epimerization at the position 2ꢁ in the proline ring via the enamine
intermediate resulting from iminium-enamine tautomerization would have
been considered as a plausible side reaction of the present reductive alkyla-
tion strategy. If this had taken place, a diastereomeric mixture consisting of
the (2ꢁR,4ꢁR) and (2ꢁS,4ꢁR) products would have formed. 1H and 13C NMR
spectra of the compounds 3 and 4 suggested the presence of only one di-
astereomer in all cases. Furthermore, HPLC analysis of the compound 4a,
which possesses (2ꢁR,4ꢁR) stereochemistry, revealed no detectable contam-
ination by the (2ꢁS,4ꢁR) diastereomer 4d (<0.5%, verified by co-injection
tR RR = 46.4 minutes, tR SR = 47.2 minutes). This confirmed that no sig-
nificant epimerization had occurred during the reductive alkylation and
deprotection steps.
The present reductive alkylation of nucleobase-derivatized proline
proved to be a more efficient route for the synthesis of aepPNA monomers
than the previously reported methods. It provided the Fmoc-protected inter-
mediates 4a–4g directly without the need for protecting group conversion.
The Fmoc-protected intermediate 3a (B1 = TBz) has previously been ob-
tained in 22% overall yield (6 steps) starting from 2a,[12] while the present
method gave the desired products in 40–89% overall yield (two steps) de-
pending on the nucleobases.
Synthesis of aepPNA Oligomers
Oligomerization of the activated aepPNA monomers was performed by
manual Fmoc-solid phase peptide synthesis methodology.[19] The sequences
synthesized included four homothymine aepPNA decamers with different
configurations at the proline moiety (denoted RR-T10, SS-T10, RS-T10 and
SR-T10 in Table 2), and a mixed base decamer with (2ꢁR,4ꢁR) configurations
(denoted RR-mix in Table 2). All sequences were capped with acetyl and
lysine amide at the N- and C-termini respectively. Generally, the average
coupling yields of 95–99% for each step were achieved according to UV ab-
sorbance measurement of the dibenzofulvene-piperidine adduct released
during deprotection step. In cases of sequences containing nucleobases
TABLE 2 Sequences and characterization details of the aepPNA synthesized
Entry
Code
Config.
Sequencea
M·H+ (calcd.)
M·H+ (found)b
1
2
3
4
5
RR-T10
SS-T10
SR-T10
RS-T10
RR-mix
(2ꢁR,4ꢁR)
(2ꢁS,4ꢁS)
(2ꢁS,4ꢁR)
(2ꢁR,4ꢁS)
(2ꢁR,4ꢁR)
TTTTTTTTTT
TTTTTTTTTT
TTTTTTTTTT
TTTTTTTTTT
GTAGATCACT
2830.05
2830.05
2830.05
2830.05
2877.09
2830.04
2830.27
2830.31
2830.24
2877.57
aN -Ac, C-LysNH2 capped.
bMALDI-TOF, α-cyano-4-hydroxy cinnamic acid (CCA) matrix.

102
P. Ngamwiriyawong and T. Vilaivan
other than thymine, the nucleobase protecting groups (Bz, Ibu) were re-
moved after completion of the synthesis by treatment of the support-bound
PNA with aqueous ammonia/dioxane 1:1 at 60◦C for 6 hours prior to the
usual TFA cleavage. The decamer aepPNAs were obtained after reverse phase
HPLC purification and were characterized by MALDI-TOF mass spectrome-
try. In all cases quasi-molecular ions were observed with m/z within 0.5 Da
to the calculated values, thus confirming the identities of all PNA synthesized
(Table 2).
DNA and RNA Binding Properties of aepPNA
Interactions between the decamer aepPNA with complementary DNA
and RNA were investigated by Tm measurement. The thermal transition
curves were recorded at 260 nm over 20–90◦C range and the Tm values are
shown in Table 3.
The binding experiments clearly indicate that the homothymine aepPNA
decamers with cis-configurations (RR-T10 and SS-T10) could form reasonably
stable hybrids with poly(rA) (Tm = 43 and 42◦C) but not poly(dA). On the
other hand, the homothymine aepPNA decamers with trans-configurations
(RS-T10 and SR-T10) failed to form stable hybrids with both poly(rA) and
poly(dA), although an apparent melting was observed between RS-T10 and
poly(dA) at a rather low temperature (24◦C). CD spectra of the aepPNA and
their mixture with their complementary DNA/RNA were in good agreement
with Tm data. Significant differences between the observed and sum of the
individual component CD spectra, which indicate hybrid formation, were ob-
served only for the pairs RR-T10/poly(rA) and SS-T10/poly(rA) (Figure 2).
Disappointingly, no hybridization of the mixed base aepPNA decamer (RR-
mix) to complementary DNA and RNA could be observed in both parallel
TABLE 3 Tm values of hybrids between decamer aepPNA and polynucleotides
Entry
aepPNA
polynucleotide
Tm (◦C)a
1
2
3
4
5
6
7
8
9
RR-T10
SS-T10
SR-T10
RS-T10
RR-T10
SS-T10
SR-T10
RS-T10
RR-mix
RR-mix
RR-mix
RR-mix
poly(rA)
poly(rA)
poly(rA)
poly(rA)
poly(dA)
poly(dA)
poly(dA)
poly(dA)
d(CATCTAGTGA)
d(AGTGATCTAC)
r(CAUCUAGUGA)
r(AGUGAUCUAC)
43
42
<20
<20
<20
<20
<20
24
<20
<20
<20
<20
10
11
12
aTm were measured at [PNA] = [DNA] = 1 µM, 10 mM sodium phosphate pH 7.0, heating rate
1◦C/min.

Synthesis and Nucleic Acids Binding Properties of aepPNA
103
FIGURE 2 CD spectra of PNA-DNA or RNA hybrids. Conditions: [PNA] = [DNA or RNA] = 1 µM,
10 mM sodium phosphate pH 7.0, 25◦C.
(Table 3, entries 9 and 11) and antiparallel orientations (Table 3, entries
10 and 12). The duplex structures formed between the mixed base aepPNA
with (2ꢁR,4ꢁR) configuration is therefore likely to be less stable than the cor-
responding DNA·DNA or DNA·RNA hybrids.[20] The unusually high stability
of the homothymine aepPNA hybrids compared to the mixed sequence in
this case could be explained by the formation of triplex structures, which was
confirmed by ultraviolet (UV) titration experiments. It had been previously
observed in other PNA systems with homopyrimidine sequences that these
triplexes could form exclusively even when the stoichiometry was not correct
because the triplex was more stable than the intermediate duplex.[1] Our
results with the (2ꢁS,4ꢁS) isomer is in accordance with the observation by
Liu that no stable hybrid was formed between homothymine aepPNA and its
complementary DNA.[11] Preliminary time-dependent UV experiments indi-
cated that the binding between the aepPNA (RR-T10 and SS-T10) and RNA
are kinetically slow. More than 30 minutes are required for the maximum
hypochromism to be established (Figure 3). The significant hysteresis ob-
served in the cooling curves of both aepPNA-RNA hybrids also confirmed the
slow kinetics of the hybridization, which may suggest that there was substan-
tial reorganization of the single stranded counterparts upon hybridization.
No hypochromism was observed in the corresponding DNA experiments,
confirming that both aepPNAs could not bind to their complementary DNA
under these conditions. Whether the preferential binding of the aepPNA to
RNA, and the failure of the mixed base aepPNA to bind to both DNA and

104
P. Ngamwiriyawong and T. Vilaivan
FIGURE 3 Time-dependent normalized UV absorbance at 268 nm of aepPNA-DNA/RNA hybridization
reactions. Conditions: [PNA] = [DNA or RNA] = 1 µM, 10 mM sodium phosphate pH 7.0, 25◦C.
RNA are due to thermodynamic or kinetic factors, and the structural basis
of this selectivity, remains to be further investigated.
CONCLUSION
The reductive alkylation of nucleobase-modified proline derivative with
Fmoc-aminoacetaldehyde (1) was found to be an efficient method for the
synthesis of aepPNA monomers. Deprotection of the C-terminal protecting
group gave the (2ꢁR,4ꢁR)-aepPNA monomers containing all four nucleobase
(ABz, T, CBz, and GIbu) and the thymine monomers with different config-
uration (2ꢁR,4ꢁR, 2ꢁS,4ꢁS, 2ꢁR,4ꢁS, 2ꢁS,4ꢁR). Oligomerization of the PNA
monomers was carried out employing Fmoc SPPS. Five aepPNA decamers
were successfully synthesized. The hybridization properties of the aepPNA
were investigated by UV and circular dichroism (CD) spectroscopy. The
results suggested that the stereochemistry on the pyrrolidine ring and the
nucleobase sequence can have a dramatic effect on the binding characteris-
tics of the aepPNA. Only the cis-stereoisomers with a homothymine sequence
can form stable triplexes with RNA, but not DNA. The hybrid formation in
these cases is kinetically slow. No hybrid formation could be observed be-
tween a mixed-base sequence (2ꢁR,4ꢁR)-aepPNA with DNA and RNA in both
antiparallel and parallel orientations.

Synthesis and Nucleic Acids Binding Properties of aepPNA
105
EXPERIMENTAL
Materials and Methods
1H and 13C spectra were recorded on a Varian Mercury-400 plus NMR
spectrometer operating at 400 MHz. MALDI-TOF mass spectra were ob-
tained in reflectron mode on an Omniflex MALDI-TOF mass spectrom-
eter (Bruker Daltonics, Germany) using α-cyano-4-hydroxy cinnamic acid
(CCA) as matrix employing 0.1% trifluoroacetic acid in acetonitrile:water
(1:2) as diluents for samples preparation. CD experiments were performed
on a JASCO Model J-810 Spectropolarimeter, Japan. The CD spectra were
recorded at 25◦C from 300 to 200 nm and averaged four times then sub-
tracted from a spectrum of the buffer under the same conditions. All
chemicals were purchased from Fluka (Switzerland), Merck (Germany), or
Aldrich (USA), and were purified as appropriate. The N -tert-butoxycarbonyl
(Boc)/diphenylmethyl (Dpm) ester protected nucleobase-modified proline
derivatives 2a–2d were synthesized from trans-4-hydroxy-L-proline according
to our previously published procedure.[18]
N-Fluoren-9-ylmethoxycarbonylaminoacetaldehyde (1)
A mixture of rac-3-amino-1,2-propanediol (0.4556 g, 5 mmol), FmocCl
(1.2935 g, 5 mmol) and NaHCO3 (0.4201 g, 5 mmol) were dissolved in 1:1
dioxane:H2O (10 mL). The reaction mixture was stirred at ambient tem-
perature overnight. The solvent was removed by rotary evaporation under
reduced pressure. The residue was diluted with 20 mL of water and extracted
with dichloromethane (3 × 30 mL) The combined organic extract was dried
over magnesium sulfate and evaporated under reduced pressure to give N -
Fmoc-3-amino-1,2-propanediol as a white solid (1.3935 g, 90%). A mixture
of this intermediate (1.2534 g, 4 mmol) and NaIO4 (1.0233 g, 4.8 mmol)
were dissolved in 1:1 MeCN:H2O (8 mL) and allowed to stir at 30◦C for
3 hours. The solvents were removed by rotary evaporation and the residue
was diluted with 20 mL of water and extracted with dichloromethane (3 ×
30 mL). The combined organic extract was dried over magnesium sulfate
and evaporated to give the product as a white fluffy solid; yield: 1.1224 g
1
(99%); m.p. 141.0–143.0◦C; H NMR (400 MHz, CDCl3) δ 4.21 (d J = 5.0
Hz, 2H), 4.27 (t J = 6.8 Hz, 1H), 4.47 (d J = 6.8 Hz, 2H), 5.48 (br m, 1H),
7.36 (m, 2H), 7.45 (m, 2H), 7.64 (d J = 7.5 Hz, 2H), 7.81 (d J = 7.5 Hz, 2H),
9.71 (s, 1H); 13C NMR (100 MHz, CDCl3) δ 47.1, 51.7, 67.2, 120.0, 125.1,
127.1, 127.8, 141.3, 143.7, 156.3, 196.5.
A Representative Procedure for the Reductive Alkylation
The protected proline derivative 2 (1 mmol) was treated with p-
toluenesulfonic acid (3 mmol) in MeCN (5 mL). The solvent was removed by

106
P. Ngamwiriyawong and T. Vilaivan
rotary evaporation at 30◦C. The residue was dissolved in MeOH (2 mL) fol-
lowed by successive addition of 1 (1.2 mmol), NaOAc (0.41 g) and NaBH3CN
(1.2 mmol). The solution was stirred at ambient temperature (30◦C) for one
hour then the solvent was removed by rotary evaporation, diluted with ethyl
acetate, and extracted with 10% HCl. The organic layer was washed with aq.
NaHCO3 and combined organic phase was dried over magnesium sulfate
and evaporated under reduced pressure. The residue was purified by flash
column chromatography eluting with hexanes:ethyl acetate (1:1) on silica
gel to afford 3.
A Representative Procedure for Deprotection of the Dpm Ester 3
A mixture of the protected intermediate 3 (0.3–0.5 mmol), anisole
(1 mL) and trifluoroacetic acid (3 mL) was stirred at ambient temperature
(30◦C) overnight. The volatiles were removed under a gentle N2 stream.
Diethyl ether (20 mL) was added to the residue to precipitate the product
4, which was collected by filtration and dried under vacuum.
Spectroscopic Data of Compounds 3a–3g and 4a–4g
N-2-(N-Fluoren-9-ylmethoxycarbonylamino)ethyl-(4ꢀR)-(N3-benzoylthymin-1-
yl)- (2ꢀR)-proline diphenylmethyl ester (3a)
1
White foam; yield: 0.6052 g (76%); H NMR (400 MHz, CDCl3) δ 1.87
(s, 3H), 2.01 (m, 1H), 2.65 (m, 1H), 2.78–2.88 (m, 2H), 2.93 (dt, J = 14.4,
9.2 Hz, 1H), 3.18 (m, 1H), 3.31 (m, 1H), 3.39 (m, 1H), 3.49 (m, 1H), 4.26
(t J = 6.8 Hz, 1H), 4.35 (dd J = 9.9, 7.4 Hz, 1H), 4.52 (dd, J = 10.2, 7.3 Hz,
1H), 5.28 (m, 1H), 5.58 (m,1H), 7.03 (s, 1H), 7.32–7.49 (m, 14H), 7.51 (t J
= 7.7 Hz, 2H), 7.64–7.73 (m, 3H), 7.82 (d J = 7.4 Hz, 2H), 7.94–8.02 (m,
3H); 13C NMR (100 MHz, CDCl3) δ 12.7, 36.6, 39.5, 47.3, 52.7, 53.3, 58.2,
65.0, 66.8, 78.2, 111.4, 120.1, 125.3, 127.0, 127.3, 127.9, 128.5, 128.8, 129.3,
130.5, 131.7, 135.2, 138.0, 139.4, 139.5, 141.4, 144.0, 150.0, 156.6, 162.9,
169.5, 172.5; MS (MALDI-TOF) m/z 797.64 [M+Na+]; HRMS (FAB+) m/z
calcd for C47H42N4O7·H+: 775.3132, found: 775.3154.
N-2-(N-Fluoren-9-ylmethoxycarbonylamino)ethyl-(4ꢀS)-(N3-benzoylthymin-1-yl)-
(2ꢀS)-proline diphenylmethyl ester (3b)
1
White foam; yield: 0.6840 g (87%); H NMR (400 MHz, CDCl3) δ 1.87
(s, 3H), 2.01 (m, 1H), 2.66 (m, 1H), 2.77–2.86 (m, 2H), 2.93 (dt, J = 14.9,
9.6 Hz, 1H), 3.18 (m, 1H), 3.31 (d J = 10.7 Hz, 1H), 3.40 (m, 1H), 3.49 (m,
1H), 4.26 (t J = 6.8 Hz, 1H), 4.36 (dd J = 10.1, 7.1 Hz, 1H), 4.53 (dd, J =
10.5, 7.3 Hz, 1H), 5.29 (m, 1H), 5.58 (m,1H), 7.04 (s, 1H), 7.33–7.47 (m,
14H), 7.52 (t J = 7.8 Hz, 2H), 7.64–7.70 (m, 3H), 7.83 (d J = 7.5 Hz, 2H),
7.95–8.00 (m, 3H); 13C NMR (100 MHz, CDCl3) δ 12.6, 36.6, 39.4, 47.3, 52.5,
53.3, 58.3, 65.0, 66.8, 78.2, 111.5, 120.1, 125.1, 126.9, 127.2, 127.8, 128.4,

Synthesis and Nucleic Acids Binding Properties of aepPNA
107
128.8, 129.2, 130.5, 131.7, 135.0, 137.6, 139.3, 139.4, 141.4, 144.0, 150.0,
156.6, 162.7, 169.2, 172.5; MS (MALDI-TOF) m/z 797.67 [M+Na+]; HRMS
(FAB+) m/z calcd for C47H42N4O7·H+: 775.3132, found: 775.3122.
N-2-(N-Fluoren-9-ylmethoxycarbonylamino)ethyl-(4ꢀS)-(N3-benzoylthymin-1-yl)-
(2ꢀR)-proline diphenylmethyl ester (3c)
1
White foam; yield: 0.3185 g (40%); H NMR (400 MHz, CDCl3) δ 2.06
(s, 3H), 2.25 (m, 1H), 2.53 (m, 1H), 2.72 (m, 2H), 2.96 (m, 1H), 3.27 (m,
2H), 3.40 (m, 1H), 4.04 (dd J = 8.2, 2.8 Hz, 1H), 4.23 (t J = 6.9 Hz, 1H),
4.43 (m, 2H), 5.25 (m, 1H), 5.48 (m, 1H), 6.95 (s, 1H), 7.27–7.46 (m, 15H),
7.49 (t J = 7.9 Hz, 2H), 7.61–7.66 (m,3H), 7.79 (d J = 7.5 Hz, 2H), 7.97 (d
J = 7.4 Hz, 2H), 13C NMR (100 MHz, CDCl3) δ 12.6, 35.4, 39.3, 47.3, 51.2,
54.3, 56.0, 63.9, 66.6, 77.6, 111.6, 120.1, 125.1, 127.0, 127.1, 127.8, 128.3,
128.7, 129.3, 130.5, 131.6, 135.1, 137.7, 139.5, 139.6, 141.3, 143.9, 149.7,
156.7, 162.9, 169.3, 171.5; MS (MALDI-TOF) m/z 797.68 [M+Na+]; HRMS
(FAB+) m/z calcd for C47H42N4O7·H+: 775.3132, found: 775.3144.
N-2-(N-Fluoren-9-ylmethoxycarbonylamino)ethyl-(4ꢀR)-(N3-benzoylthymin-
1-yl)- (2ꢀS)-proline diphenylmethyl ester (3d)
White foam; yield: 0.1652 g (43%); 1H NMR (400 MHz, CDCl3) δ 2.08 (s,
3H), 2.28 (m, 1H), 2.56 (m, 1H), 2.74 (m, 2H), 2.98 (m, 1H), 3.29 (m, 2H),
3.44 (m, 1H), 4.04 (dd J = 8.2, 2.8 Hz, 1H), 4.24 (t J = 6.7 Hz, 1H), 4.44 (d
J = 6.8 Hz, 2H), 5.25 (m, 1H), 6.95 (s, 1H), 7.27–7.44 (m, 14H), 7.51 (t J =
7.8 Hz, 2H), 7.61–7.69 (m,3H), 7.80 (d J = 7.5 Hz, 2H), 7.96 (d J = 7.4 Hz,
2H), 13C NMR (100 MHz, CDCl3) δ 12.6, 35.5, 39.3, 47.3, 51.2, 54.3, 56.0,
63.9, 66.6, 77.6, 111.7, 120.1, 125.0, 127.1, 127.2, 127.8, 128.3, 128.7, 129.3,
130.5, 131.5, 135.2, 137.5, 139.5, 139.6, 141.3, 143.9, 149.7, 156.6, 162.8,
169.2, 171.5; MS (MALDI-TOF) m/z 797.80 [M+Na+]; HRMS (FAB+) m/z
calcd for C47H42N4O7·H+: 775.3132, found: 775.3138.
N-2-(N-Fluoren-9-ylmethoxycarbonylamino)ethyl-(4ꢀR)-(N2-isobutyrylguanin-
9-yl)-(2ꢀR)-proline diphenylmethyl ester (3e)
1
White foam; yield: 0.2654 g (69%); H NMR (400 MHz, CDCl3) δ 1.24
(d J = 6.0 Hz, 6H), 2.10–2.22 (m, 1H), 2.55–2.92 (m, 5H), 3.20 (m, 1H),
3.26–3.40 (m, 1H), 3.52 (m, 1H), 4.23 (t J = 6.8 Hz, 1H), 4.35 (d J = 6.8
Hz, 2H), 5.00 (m, 1H), 5.78 and 6.20 (2 × m, 1H), 6.92 (s, 1H), 7.21–7.44
(m, 15H), 7.62 (d J = 7.2 Hz, 2H), 7.75 (d J = 7.2 Hz, 2H), 7.83 and 8.14 (2
× s, 1H), 9.82 and 10.53 (2 × s, 1H), 12.12 and 12.46 (2 × s, 1H); 13C NMR
(100 MHz, CDCl3) δ 19.1, 19.2, 36.0, 36.9, 39.6, 47.2, 51.9, 53.5, 59.1, 64.7,
66.9, 77.9, 119.9, 120.2, 125.2, 126.9, 127.1, 127.7, 128.3, 128.7, 138.2, 139.4,
141.2, 144.0, 147.9, 148.5, 156.0, 156.8, 172.4, 180.0; MS (MALDI–TOF) m/z
766.86 [M+H+]; HRMS (FAB+) m/z calcd for C44H43N7O6·H+: 766.3353,
found: 766.3378.

108
P. Ngamwiriyawong and T. Vilaivan
N-2-(N-Fluoren-9-ylmethoxycarbonylamino)ethyl-(4ꢀR)-4ꢀ-(N6-benzoyladenin-
9-yl)-(2ꢀR)-proline diphenylmethyl ester (3f)
1
White foam; yield: 0.3499 g (60%); H NMR (400 MHz, CDCl3) δ 2.25
(m, 1H), 2.80 (m, 1H), 2.89 (m, 1H), 3.02 (m, 2H), 3.16 (m, 1H), 3.39 (m,
1H), 3.47 (m, 1H), 3.62 (m, 1H), 4.29 (t J = 7.2 Hz, 1H), 4.39 (m, 2H), 5.37
(m, 1H), 5.61 (m, 1H), 6.97 (s, 1H), 7.22–7.41 (m, 14H), 7.50 (t J = 7.6 Hz,
2H), 7.58–7.70 (m, 3H), 7.77 (d J = 7.2 Hz, 2H), 8.02 (d J = 7.6 Hz, 2H), 8.65
(s, 1H), 8.82 (s, 1H), 9.36 (s, 1H); 13C NMR (100 MHz, CDCl3) δ 37.2, 39.5,
47.3, 52.1, 53.6, 59.1, 64.5, 66.9, 78.1, 119.9, 122.4, 125.3, 126.9, 127.1, 127.7,
127.9, 128.3, 128.7, 128.8, 132.7, 133.8, 139.3, 141.3, 142.2, 144.1, 149.4,
151.5, 152.4, 156.6, 164.7, 172.5; MS (MALDI–TOF) m/z 784.53 [M+H+];
HRMS (FAB+) m/z calcd for C47H41N7O5·H+: 784.3247, found: 784.3271.
N-2-(N-Fluoren-9-ylmethoxycarbonylamino)ethyl-(4ꢀR)-(N4-benzoylcytosin-
1-yl)-(2ꢀR)-proline diphenylmethyl ester (3g)
1
White foam; yield: 0.3231 g (89%); H NMR (400 MHz, CDCl3) δ 2.05
(m, 1H), 2.67 (m, 1H), 2.86 (m, 2H), 2.98 (m, 1H), 3.15 (m, 1H), 3.34 (m,
1H), 3.41 (m, 1H), 3.51 (m, 1H), 4.28 (t J = 7.2 Hz, 1H), 4.42 (t J = 6.8
Hz, 2H), 5.43 (m, 1H), 6.96 (s, 1H), 7.18–7.72 (m, 14H), 7.42 (m, 2H), 7.53
(m, 2H), 7.61–7.72 (m, 2H), 7.79 (d J = 7.2 Hz, 2H), 7.93 (m, 2H), 8.43 (d
J = 6.4 Hz, 1H); 13C NMR (400 MHz, CDCl3) δ 36.6, 39.4, 47.2, 53.7, 54.3,
58.6, 65.2, 66.8, 78.1, 97.5, 119.9, 125.3, 127.7, 128.3, 126.9, 127.1, 127.7,
127.8, 128.4, 128.8, 128.9, 133.1, 139.3, 141.3, 144.1, 146.8, 156.0, 156.6,
161.9, 166.5, 172.4; HRMS (FAB+) m/z calcd for C46H41N5O6·H+: 760.3135,
found: 760.3137.
N-2-(N-Fluoren-9-ylmethoxycarbonylamino)ethyl-(4ꢀR)-(thymin-1-yl)-
(2ꢀR)-proline (4a)
1
White solid; yield: 0.2545 g (85%); H NMR (400 MHz, DMSO–d6 + 1
drop TFA) δ 1.77 (s, 3H), 2.35 (m, 1H), 2.83 (m, 1H), 3.20 (m, 1H), 3.37
(m, 2H), 3.51 (m, 1H), 3.69 (m, 1H), 3.94 (m, 1H), 4.26 (t J = 6.8 Hz, 1H),
4.40 (m, 2H), 4.54 (m, 1H), 5.14 (m, 1H), 7.33 (t J = 7.2 Hz, 2H), 7.41 (t J
= 7.2 Hz, 2H), 7.57 (s, 1H), 7.69 (d J = 7.2 Hz, 2H), 7.89 (d J = 7.2 Hz, 2H);
13C NMR (100 MHz, DMSO–d6 + 1 drop TFA) δ 12.7, 34.4, 38.3, 47.2, 53.3,
54.2, 57.5, 66.0, 66.2, 109.7, 120.6, 125.6, 127.6, 128.1, 139.3, 141.2, 144.3,
151.6, 156.7, 164.2, 171.5; MS (MALDI–TOF) m/z 505.13 [M+H+]; HRMS
(FAB+) m/z calcd for C27H28N4O6·H+: 505.2087, found: 505.2102.
N-2-(N-Fluoren-9-ylmethoxycarbonylamino)ethyl-(4ꢀS)-(thymin-1-yl)-
(2ꢀS)-proline (4b)
1
White solid; yield: 0.3711 g (72%); H NMR (400 MHz, DMSO–d6 + 1
drop TFA) δ 1.76 (s, 3H), 2.37 (m, 1H), 2.83 (m, 1H), 3.20 (m, 1H), 3.37
(m, 2H), 3.52 (m, 1H), 3.69 (dd J = 12.5, 10.1 Hz, 1H), 3.95 (dd J = 12.6,

Synthesis and Nucleic Acids Binding Properties of aepPNA
109
3.9 Hz, 1H), 4.25 (t J = 6.4 Hz, 1H), 4.36–4.44 (m, 2H), 4.54 (m, 1H), 5.15
(m, 1H), 7.32 (t J = 7.3 Hz, 2H), 7.40 (t J = 7.3 Hz, 2H), 7.56 (s, 1H),
7.68 (d J = 7.5 Hz, 2H), 7.87 (d J = 7.5 Hz, 2H); 13C NMR (100 MHz,
DMSO–d6 + 1 drop TFA) δ 12.7, 34.2, 38.1, 47.2, 53.4, 54.3, 57.4, 66.0, 66.3,
109.8, 120.6, 125.6, 127.6, 128.1, 139.2, 141.2, 144.3, 151.6, 156.7, 164.3,
171.1; MS (MALDI–TOF) m/z 505.13 [M+H+]; HRMS (FAB+) m/z calcd
for C27H28N4O6·H+: 505.2087, found: 505.2088.
N-2-(N-Fluoren-9-ylmethoxycarbonylamino)ethyl-(4ꢀS)-(thymin-1-yl)-
(2ꢀR)-proline (4c)
1
White solid; yield: 0.1201 g (72%); H NMR (400 MHz, DMSO–d6 + 1
drop TFA) δ 1.78 (s, 3H), 2.49 (m, 1H), 2.66 (m, 1H), 3.25 (m, 1H), 3.38
(m, 2H), 3.47 (m, 1H), 3.58 (m, 1H), 4.00 (dd J = 11.3, 8.8 Hz, 1H), 4.25
(t J = 6.3 Hz, 1H), 4.40 (d J = 6.6 Hz, 2H), 4.77 (t J = 9.5 Hz, 1H), 4.98
(m, 1H), 7.33 (t J = 7.3 Hz, 2H), 7.42 (t J = 7.4 Hz, 2H), 7.54 (br m, 1H),
7.62 (s, 1H), 7.70 (d J = 7.4 Hz, 2H), 7.90 (d J = 7.5 Hz, 2H); 13C NMR
(100 MHz, DMSO–d6 + 1 drop TFA) δ 12.6, 33.6, 38.6, 47.2, 52.9, 53.0, 55.6,
65.1, 65.9, 110.0, 120.6, 125.6, 127.5, 128.1, 138.8, 141.2, 144.3, 151.3, 156.7,
164.3, 172.3; MS (MALDI–TOF) m/z 505.12 [M+H+]; HRMS (FAB+) m/z
calcd for C27H28N4O6·H+: 505.2087, found: 505.2062.
N-2-(N-Fluoren-9-ylmethoxycarbonylamino)ethyl-(4ꢀR)-(thymin-1-yl)-
(2ꢀS)-proline (4d)
1
White solid; yield: 0.1201 g (72%); H NMR (400 MHz, DMSO–d6 + 1
drop TFA) δ 1.78 (s, 3H), 2.46 (m, 1H), 2.67 (m, 1H), 3.26 (m, 1H), 3.38
(m, 2H), 3.48 (m, 1H), 3.58 (m, 1H), 4.01 (dd J = 10.8, 8.9 Hz, 1H), 4.25
(t J = 6.4 Hz, 1H), 4.40 (d J = 6.7 Hz, 2H), 4.78 (t J = 9.3 Hz, 1H), 4.98
(m, 1H), 7.33 (t J = 7.3 Hz, 2H), 7.41 (t J = 7.4 Hz, 2H), 7.61 (br s, 1H),
7.52 (br m, 1H), 7.69 (d J = 7.3 Hz, 2H), 7.89 (d J = 7.5 Hz, 2H); 13C NMR
(100 MHz, DMSO–d6 + 1 drop TFA) δ 12.6, 33.8, 38.7, 47.2, 52.8, 53.0, 55.7,
65.0, 65.9, 110.0, 120.6, 125.6, 127.5, 128.1, 138.7, 141.2, 144.3, 151.3, 156.7,
164.3, 172.7; MS (MALDI–TOF) m/z 505.00 [M+H+]; HRMS (FAB+) m/z
calcd for C27H28N4O6·H+: 505.2087, found: 505.2063.
N-2-(N-Fluoren-9-ylmethoxycarbonylamino)ethyl-(4ꢀR)-(N2-isobutyrylguanin-
9-yl)-(2ꢀR)-proline (4e)
1
White solid; yield: 0.1565 g (77%); H NMR (400 MHz, DMSO–d6 + 1
drop TFA) δ 1.14 (d, J = 6.8 Hz, 6H), 2.20 (m, 1H), 2.70 (m, 1H), 2.79
(sept J = 6.8 Hz, 1H), 2.90 (m, 1H), 2.98–3.13 (m, 2H), 3.17–3.29 (m,
2H), 3.50–3.67 (m, 2H), 4.20–4.43 (m, 3H), 5.04 (s, 1H), 7.34 (m, 2H),
7.42 (m, 2H), 7.70 (d J = 7.2 Hz, 2H), 7.89 (d J = 7.2 Hz, 2H), 8.26 (s,
1H), 11.67 (s, 1H), 12.08 (s, 1H); 13C NMR (100 MHz, DMSO–d6 + 1 drop
TFA) δ 19.3, 35.2, 36.2, 39.1, 47.2, 52.1, 54.0, 58.7, 65.4, 65.9, 120.1, 120.6,

110
P. Ngamwiriyawong and T. Vilaivan
125.6, 127.6, 128.1, 138.5, 141.2, 144.4, 148.2, 148.7, 155.4, 156.6, 173.5,
180.5; MS (MALDI–TOF) m/z 600.19 [M+H+]; HRMS (FAB+) m/z calcd
for C31H33N7O6·H+: 600.2571, found: 600.2568.
N-2-(N-Fluoren-9-ylmethoxycarbonylamino)ethyl-(4ꢀR)-(N6-benzoyladenin-9
-yl)- (2ꢀR)-proline (4f)
1
White solid; yield: 0.1431 g (75%); H NMR (400 MHz, DMSO–d6 + 1
drop TFA) δ 2.34 (m, 1H), 2.84 (m, 1H), 2.90–3.08 (m, 2H), 3.17–3.39 (m,
3H), 3.75 (m, 1H), 3.88 (m, 1H), 4.21–4.30 (m, 3H), 5.46 (m, 1H), 7.22–7.41
(m, 4H), 7.46 (br m, 1H), 7.57 (t J = 7.6 Hz, 2H), 7.52–7.74 (m, 3H), 7.89
(d J = 7.6 Hz, 2H), 8.08 (d J = 7.6 Hz, 2H), 8.72 (s, 1H), 8.75 (s, 1H); 13
C
NMR (100 MHz, DMSO–d6 + 1 drop TFA) δ 35.7, 38.7, 47.1, 52.2, 54.1, 58.4,
65.7, 66.0, 120.6, 125.6, 125.7, 127.6, 128.1, 128.9, 129.0, 133.0, 133.8, 141.2,
143.7, 144.3, 150.7, 151.6, 152.3, 156.7, 166.2, 172.5; MS (MALDI–TOF) m/z
618.19 [M+H+]; HRMS (FAB+) m/z calcd for C34H31N7O5·H+: 618.2465,
found: 618.2466.
N-2-(N-Fluoren-9-ylmethoxycarbonylamino)ethyl-(4ꢀR)-(N4-benzoylcytosin-1-
yl)- (2ꢀR)-proline (4g)
1
White solid; yield: 0.2226 g (74%); H NMR (400 MHz, DMSO–d6 + 1
drop TFA) δ 2.47 (m, 1H), 2.98 (m, 1H), 3.24 (m, 1H), 3.41 (m, 2H), 3.57
(m, 1H), 3.77 (m, 1H), 4.14 (m, 1H), 4.28 (m, 1H), 4.36 (m, 2H), 4.60 (m,
1H), 5.13 (m, 1H), 7.32 (m, 2H), 7.42 (m, 2H), 7.54 (m, 2H), 7.58–7.74
(m, 4H), 7.90 (d J = 6.4 Hz, 2H), 8.00 (d J = 7.6 Hz, 2H), 8.20 (d J = 5.6
Hz, 1H); 13C NMR (400 MHz, DMSO–d6) δ 35.6, 39.0, 47.2, 53.9, 55.8, 58.0,
65.9, 66.0, 96.8, 120.6, 125.6, 127.5, 128.1, 128.9, 129.5, 133.2, 133.7, 141.2,
144.3, 148.6, 155.7, 156.7, 163.2, 167.9, 173.0; MS (MALDI–TOF) m/z 594.11
[M+H+]; HRMS (FAB+) m/z calcd for C33H31N5O6·H+: 594.2353, found:
594.2334.
Solid Phase Synthesis of aepPNA Oligomers
The Fmoc-protected PNA monomers 4a–4g were activated just before use
as pentafluorophenyl esters by treatment with 1 equiv of pentafluorophenyl
trifluoroacetate and DIEA. The products obtained following acid-base ex-
traction to remove DIEA salts were unstable upon prolonged storage and
thus were used without further purification. All peptide syntheses were car-
ried out manually in a custom-made glass column with a fritted glass tip
(i.d. = 5 mm). The reaction column was filled with TentaGel S RAM Fmoc
resin (0.24 meq/g loading, 6.3 mg, 1.5 µmol). The resin, after swollen in
DMF, was deprotected by treating with 20% piperidine in DMF (500 µL) for
15 minutes at room temperature with occasional agitation. After washing,
the deprotected resin was treated with Fmoc-L-Lys(Boc)-OPfp (9.5 mg, 15
µmol) and HOAt (2.0 mg, 15 µmol) dissolved in anhydrous DMF (35 µL).

Synthesis and Nucleic Acids Binding Properties of aepPNA
111
After 2 hours at room temperature, the resin was washed exhaustively with
DMF and capped with acetic anhydride/DIEA mixture (Ac2O 10 µL, DIEA
10 µL and DMF 80 µL). Next the Fmoc deprotection was repeated and the
resin was treated with an appropriate activated PNA monomer (6.0 µmol)
and 7-aza-1-hydroxybenzotriazole (HOAt) (0.8 mg, 6.0 µmol) dissolved in
35 µL DMF for 2 hours. After capping, the deprotection-coupling-capping
steps were repeated until the desired sequence was obtained. After final
cleavage of the Fmoc protecting group, the PNA was end-capped by acetyla-
tion with 10% Ac2O/DIEA in DMF. With the exception of homothymine se-
quence, the resin-bound aepPNA was treated with 1:1 concentrated aqueous
ammonia-dioxane at 60◦C overnight to remove the exocyclic amino protect-
ing groups. The crude aepPNA was released from the resin by treatment with
trifluoroacetic acid (1 mL) at room temperature for 1 hour with occasional
agitation. The trifluoroacetic acid was removed by a nitrogen stream (fume
hood). The procedure was repeated two more times to ensure a complete
cleavage of the peptide from the resin. The crude aepPNA was centrifugally
washed with diethyl ether (3 times) and was air-dried at room temperature.
Analysis and purification was performed by reverse phase HPLC on a Water
600TM system equipped with gradient pump and Water 996 photodiode array
detector. A Hypersil C18 HPLC column (4.6 × 250 mm, 5 µ particle size)
was used for both analytical and preparative purposes. Elution was carried
out with a gradient system of 0.1% TFA in acetonitrile/water and moni-
tored at 260 nm. The purity and identity of the products were confirmed by
MALDI-TOF mass spectrometry.
Tm Experiments
Tm experiments were performed on a CARY 100 Bio UV-Visible spec-
trophotometer (Varian Ltd., Australia) equipped with a thermal melt sys-
tem. The sample for Tm measurement was prepared by mixing calculated
amounts of stock oligonucleotide and aepPNA solutions together to give fi-
nal concentration of nucleotides and sodium phosphate buffer (pH 7.0) and
the final volumes were adjusted to 3.0 mL by addition of deionized water.
The samples were transferred to a 10 mm quartz cell with a Teflon stopper
and equilibrated at the starting temperature for 10 minutes. The A260 was
recorded in steps from 20–90◦C (block temperature) with a temperature
increment of 1◦C/min. The temperature was corrected by applying a lin-
ear equation obtained from temperature probe read out. The results were
normalized by dividing the absorbance at each temperature by the initial
absorbance. Tm values were obtained from first derivative plots.
REFERENCES
1. Uhlmann, E.; Peyman, A.; Breipohl, G.; Will, D. W. PNA: Synthetic polyamide nucleic acids with
unusual binding properties. Angew. Chem. Int. Ed. 1998, 37, 2796–2823.

112
P. Ngamwiriyawong and T. Vilaivan
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