DNA-, RNA- and self-pairing properties of a pyrrolidinyl peptide nucleic acid with a (2′R,4′S)-prolyl-(1S,2S)-2-aminocyclopentanecarboxylic acid backbone

Article abstract of DOI:10.1016/j.tetlet.2010.08.102

A new pyrrolidinyl peptide nucleic acid (PNA) comprising of an alternate sequence of 4′-nucleobase-modified proline with (2′R,4′S) configuration and a (1S,2S)-2-aminocyclopentanecarboxylic acid [(2′R,4′S)-acpcPNA] backbone was synthesized and its DNA-, RN

Full text of DOI:10.1016/j.tetlet.2010.08.102

Tetrahedron Letters 51 (2010) 5822–5826
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Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
DNA-, RNA- and self-pairing properties of a pyrrolidinyl peptide nucleic
acid with a (20R,40S)-prolyl-(1S,2S)-2-aminocyclopentanecarboxylic acid
backbone
Jaru Taechalertpaisarn, Pitchanun Sriwarom, Chalotorn Boonlua, Nattawut Yotapan, Chotima Vilaivan,
⇑
Tirayut Vilaivan
Organic Synthesis Research Unit, Department of Chemistry, Faculty of Science, Chulalongkorn University, Phayathai Road, Patumwan, Bangkok 10330, Thailand
a r t i c l e i n f o
a b s t r a c t
A new pyrrolidinyl peptide nucleic acid (PNA) comprising of an alternate sequence of 40-nucleobase-
Article history:
modified proline with (20R,40S) configuration and
a (1S,2S)-2-aminocyclopentanecarboxylic acid
Received 13 May 2010
Revised 6 August 2010
Accepted 31 August 2010
Available online 6 September 2010
[(20R,40S)-acpcPNA] backbone was synthesized and its DNA-, RNA- and self-pairing properties studied.
Tm and CD studies suggested that the (20R,40S)-acpcPNA forms antiparallel hybrids to DNA and RNA with
high sequence and direction specificity. The stability of these hybrids is comparable to those of the
(20R,40R)-acpcPNA hybrids previously reported by our group. On the other hand, experiments with a
self-complementary sequence indicated that the new (20R,40S)-acpcPNA forms a more stable antiparallel
self-hybrid than (20R,40R)-acpcPNA.
Keywords:
b-Amino acid
DNA
Ó 2010 Elsevier Ltd. All rights reserved.
PNA
Diastereomer
Epimer
Foldamer
a-DNA
A peptide nucleic acid (PNA) is a DNA analogue that binds with
(20R,40R)-acpcPNA (Scheme 1), can form particularly stable antipar-
allel hybrids with DNA and less so with RNA.7d On the other hand,
the effect of changing the configuration of the pyrrolidine in these
b-amino acid-containing PNAs has not been systemically
investigated.
single- and double-stranded nucleic acids with high affinity and se-
quence specificity.1 In addition to their stronger binding affinity,
they are chemically and physiologically more stable than DNA/
RNA. These properties lead to a number of applications in the field
of diagnostics, therapeutics and biotechnology.2 After almost two
decades since the first peptide nucleic acid (PNA) was introduced
by Nielsen et al.3 this original PNA system (also known as aegPNA)
is still perhaps the only PNA system to have found widespread use.
Many attempts have been made to improve current PNA technology
by incorporating rigidity to the flexible aegPNA backbone in such a
way that the PNA backbone is pre-organized for nucleic acid bind-
ing.4,5 Inspired by the concept of foldamers,6 we recently introduced
a series of novel, conformationally rigid PNA analogues with a pyr-
rolidine core structure in combination with b-amino acid spacers.7
Preliminary screening based on the (20R,40R) stereoisomer of the
nucleobase-modified proline at the 40 position, which corresponded
to the configuration of natural nucleosides, revealed only certain
allowable b-amino acid spacers. In particular, the PNA carrying
It is well known that a-anomeric DNA and its backbone-modi-
fied analogues can bind to normal b-DNA, albeit with polarity
reversal (the two strands must be parallel to each other).8–10 We
therefore proposed that the pyrrolidinyl PNA epimer with a
(20R,40S)-prolyl-(1S,2S)-2-aminocyclopentanecarboxylic acid back-
bone, that is, (20R,40S)-acpcPNA (Scheme 1), should retain the abil-
ity to bind to the target DNA. Furthermore, this epimeric acpcPNA
might exhibit different directional specificity (parallel vs antiparal-
lel) and preference for binding to different types of nucleic acid
targets (DNA vs RNA) as observed with
a
-anomeric DNA.11 Preli-
minary UV-melting experiments with the remaining three diaste-
reomeric pyrrolidinyl PNAs with the same T5 sequence and
(20S,40S), (20S,40R) and (20R,40S) configurations of the proline moiety
revealed that only the (20R,40S) diastereomer binds to poly(dA). To
investigate the binding behaviour of this particular epimeric PNA
in detail, studies of more complex sequences are required.
D
-aminopyrrolidinecarboxylic acid (DAPC)7a,b or (1S,2S)-2-amino-
cyclopentanecarboxylic acid (ssACPC)7c,d spacers, for example,
The four nucleobase-modified pyrrolidine monomers (A, T, C
and G) were prepared starting from the known intermediate 112
according to the method outlined in Scheme 2. In brief, the nucle-
obases were installed on the pyrrolidine ring, with inversion of
⇑
Corresponding author. Tel.: +66 2 2187627; fax: +66 2 2187598.
E-mail address: vtirayut@chula.ac.th (T. Vilaivan).
0040-4039/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2010.08.102

J. Taechalertpaisarn et al. / Tetrahedron Letters 51 (2010) 5822–5826
5823
O
O
O
O
R
R
B
B
B
R
S
B
N
N
O
O
O
O
H
H
O
O
P
P
N
N
O
O
O
O
n
n
n
n
(2'R,4'S)-acpcPNA
(2'R,4'R)-acpcPNA
Scheme 1.
DNA
α-DNA
TBz
HO2C
T
iii)
DpmO2C
OH
ii)
DpmO2C
N
N
48%
N
46%
Fmoc
Boc
Boc
1a
2a
3a
i)
83%
CBz
DpmO2C
CBz
DpmO2C
OTs
v)
HO2C
N
iv)
N
N
33%
83%
Boc
Boc
Fmoc
2b
3b
1b
vi)
72%
ABz
DpmO2C
A
DpmO2C
ABz
v)
HO2C
N
vii)
viii)
49%
N
56%
N
82%
Fmoc
Boc
Boc
2e
2c
3c
DpmO2C
B
DpmO2C
G
HO2C
N
GIbu
x)
ix)
N
N
57%
37%
Boc
Fmoc
Fmoc
2f
B = 2-chloro-6-aminopurin-9-yl
2d
3d
i) TsCl, Et3N, cat. DMAP, CH2Cl2, rt, 4 h
ii) N3-BzT, Ph3P, DIAD, THF, 0 oC-rt, O/N
vi) adenine, K2CO3, DMF, 80 oC, 6 h
vii) a) BzCl, pyridine, 0 oC, 2 h b) aq. NH3, MeOH, rt
viii) 2-amino-6-chloropurine, K2CO3, DMF, 80 oC, 2 h
ix) a) TFA, rt, 2 h b) 2M HCl, reflux, 2 h
iii) a) TsOH, MeCN, rt, 3 h b) FmocCl, DIEA, rt, 1 h
c) TFA, anisole, rt, O/N
iv) N4-BzC, K2CO3, DMF, 80 oC, 8 h
v) a) TFA, anisole, rt, 3 h
c) FmocOSu, NaHCO3, aq. MeCN, rt, O/N
d) Ph2CHN2, EtOAc-MeOH, rt, 2 h
x) a) (CH3)2CHCOCl, pyridine, 0 oC-rt, O/N b) TFA, 1 h
b) FmocOSu, NaHCO3, aq. MeCN, O/N
Scheme 2.
Table 1
configuration, via Misunobu reaction on 1a (T monomer) or SN2
substitution of the corresponding tosylate 1b (CBz, ABz and GIbu
monomers; Bz = benzoyl and Ibu = isobutyryl).12 Due to the poor
yield and regioselectivity associated with direct tosyl displacement
with ABz and GIbu, the displacements were carried out with adenine
and 2-amino-6-chloropurine instead. The initial products, 2e and
2f, were subsequently transformed into the desired protected
monomers by standard nucleoside chemistry. Protecting group ex-
change from N-Boc to N-Fmoc afforded the Fmoc-protected mono-
mers 3a–d for the synthesis of the PNA by Fmoc-SPPS.13 These
monomers, together with Fmoc-(1S,2S)-2-aminocyclopentanecarb-
oxylic acid14 were coupled on Tentagel resin equipped with an
acid-labile Rink amide linker via their pentafluorophenyl esters,15
as previously described for similar PNAs.7,16 One homopyrimidine
(P1) and two mixed-base PNAs (non self-complementary P2 and
self-complementary P3) were synthesized. The identities of all
the synthesized PNAs were confirmed by MALDI-TOF mass spec-
trometry (Table 1, see also Fig. S19–S21, Supplementary data) after
Sequences and characterization data of (20R,40S)-acpcPNA P1–P3
PNA
Sequencea (N ? C)
Length (bases)
m/z (calcd)b
m/z (found)c
P1
P2
P3
TTTTTTTTT
GTAGATCACT
CGCGAATTCGCG
9
10
12
3266.153
3620.907
4233.184
3265.575
3618.854
4234.538
a
All PNAs were modified with lysinamide at the C-termini and end-capped by
acetylation (P3), benzoylation (P2) or with lysine (P1).
b
Average mass, calculated for MꢁH+
c
MALDI-TOF, linear mode, a-cyano-4-hydroxycinnamic acid (CCA) matrix
cleavage from the resin using trifluoroacetic acid (TFA) and reverse
phase HPLC purification.
The interaction of the 9mer homothymine (20R,40S)-acpcPNA P1
with its complementary DNA (dA9) was first studied by UV-melt-
ing experiments. A single-transition melting curve was observed
with a Tm of 67.8 °C.17 Although this Tm value was ꢀ5 °C lower than

5824
J. Taechalertpaisarn et al. / Tetrahedron Letters 51 (2010) 5822–5826
Table 2
Tm values of hybrids between P2 and DNA/RNA
a
a
Entry
DNA/RNA sequence (50?30)
Tm (°C) (20R,40R)
Tm (°C) (20R,40S)
Note
1
2
3
4
5
6
7
dAGTGATCTAC
dAGTGCTCTAC
dAGTGTTCTAC
dAGTGGTCTAC
dCATCTAGTGA
rAGUGAUCUAC
rCAUCUAGUGA
53.3
23.8
29.4
23.9
<20
50.8
26.5
27.4
28.1
<20
Antiparallel, perfect matched
Antiparallel, single mismatched
Antiparallel, single mismatched
Antiparallel, single mismatched
Parallel, perfect matched
42.3
<20
40.7
<20
Antiparallel, perfect matched
Parallel, perfect matched
a
Conditions: 10 mM sodium phosphate buffer, pH 7.0, 100 mM NaCl; concentration of PNA = DNA = 1
0.5 °C.
lM, heating rate 1 °C/min. The Tm figures are accurate to within
a hybrid with each other.7d The absence of any melting upon heat-
ing (20R,40R)-PNA with the same sequence as P3, without its DNA
target, supported our earlier findings. On the other hand, the
self-complementary (20R,40S)-acpcPNA P3 showed a well-defined
melting curve with a Tm value of 47.8 °C suggesting the formation
of a PNAꢁPNA homoduplex (Fig. 1).18 In the presence of comple-
mentary DNA, both PNAs form very stable PNAꢁDNA heterodu-
plexes with Tm values of 77.0 °C (20R,40R) and 78.6 °C (20R,40S),
respectively. CD spectra (Fig. 2) of the self-complementary PNAs
also revealed that the self-complementary (20R,40S)-acpcPNA was
more structurally organized than (20R,40R)-acpcPNA with the same
sequence, which was consistent with the Tm results. In the pres-
ence of the DNA strand, both PNAs become more ordered as shown
by the increase in magnitude of the negative CD bands at 208 and
250 nm. The CD spectra also suggested that the helical senses of
PNAꢁDNA hybrids derived from both epimeric PNAs should be
similar.
1.02
1.00
0.98
0.96
0.94
0.92
0.90
0.88
0.86
(2'R,4'R)-PNA-PNA
(2'R,4'R)-PNA-DNA
(2'R,4'S)-PNA-PNA
(2'R,4'S)-PNA-DNA
Although only a few other workers have described the synthe-
ses and compared DNA-pairing properties of related diastereo-
meric pyrrolidinyl PNA systems,19 these comparisons were
limited to homopurine/pyrimidine sequences or chimeric back-
bones consisting of different type of PNA monomers. The present
work is the first example of critical evaluation on mixed sequences
with homogeneous, non-chimeric backbones. The exclusive anti-
parallel binding mode of (20R,40S)-acpcPNA to its complementary
DNA is similar to (20R,40R)-acpcPNA,7d and is in sharp contrast with
20
30
40
50
T (oC)
60
70
80
Figure 1. Melting curves of self-complementary PNA P3 in the absence and
presence of complementary DNA. The experiments were carried out at a concen-
tration of PNA strand = 2
M) in 10 mM sodium phosphate buffer (pH 7.0) containing 100 mM of NaCl,
heating rate = 1 °C/min.
lM (without DNA target) or 1 lM (with DNA target at
1
l
the DNA counterparts. In addition, while normal DNA and
a-ano-
that of diastereomeric (20R,40R)-acpcPNA under similar conditions
(72.9 °C), we were pleased to find that the (20R,40S)-acpcPNA could
indeed retain strong binding to DNA. The Tm values of the hybrids
of the mixed-base 10mer (20R,40S)-acpcPNA P2 and DNA were also
measured, and the data compared with (20R,40R)-acpcPNA with the
same sequence (Table 2, see also Fig. S22). In this particular se-
quence, the thermal stability of the complementary antiparallel
(20R,40S)-acpcPNAꢁDNA hybrid was only slightly lower (ꢀ2.5 °C)
than that of the corresponding (20R,40R)-acpcPNAꢁDNA hybrid
(Table 2, entry 1). No melting could be observed for the corre-
sponding complementary parallel hybrids in both cases, suggesting
a strong preference of both PNA diastereomers to bind to their DNA
targets in an antiparallel fashion (Table 2, entry 5). When a mis-
matched base was introduced to the DNA strand, the stabilities
of the mismatched PNA–DNA hybrids decreased sharply (Table 2,
entries 2–4). The Tm decrease for each (20R,40S)- and (20R,40R)-
acpcPNA mismatched hybrid was >20 °C, indicating the high
specificity in DNA recognition of both PNA systems. As previously
observed with the (20R,40R)-acpcPNA system,7d (20R,40S)-PNA also
binds with its complementary RNA, selectively, in antiparallel fash-
ion, and with a lower affinity compared to the corresponding DNA
hybrids (Table 2, entries 6 and 7).
meric DNA bind more strongly to RNA than DNA,11 the correspond-
ing epimeric (20R,40S)-acpcPNA exhibited stronger binding to DNA
over RNA being similar to (20R,40R)-acpcPNA.7d These, together
with the relatively strong, yet highly sequence specific, binding
of the new (20R,40S)-acpcPNA to DNA and RNA indicates that the
configuration of the proline ring in the pyrrolidinyl PNA can
tolerate modification to some extent, without significantly altering
the DNA-binding properties. From this study, the only significant
difference between the two epimeric PNA systems is that
(20R,40S)-acpcPNA can form a relatively stable antiparallel self-
complementary hybrid while (20R,40R)-acpcPNA cannot. This
emphasizes the delicate effect of stereochemistry on the self-pair-
ing properties of these pyrrolidinyl PNAs. Although the structural
basis of this difference is not clear at present, the explanation for
the inability of (20R,40R)-acpcPNA to self-hybridize based on steric
effects alone may not be sufficient.
This work suggests that the more easily synthesized (20R,40S)-
acpcPNA could substitute (20R,40R)-acpcPNA as probes for nucleic
acid detection20 due to their similar DNA- and RNA-binding
properties. The unexpected possibility to modulate the self-pairing
behaviour of pyrrolidinyl PNA by changing the configuration of the
pyrrolidine ring should further expand the potential scope of
applications of these pyrrolidinyl PNAs. Although the inability to
form a self-pairing hybrid of the original (20R,40R)-acpcPNA system
is highly desirable for certain applications such as DNA targeting
Finally, the two epimeric PNAs were compared in terms of the
ability to self-hybridize. It was established from our previous work
that two complementary strands of (20R,40R)-acpcPNA cannot form

J. Taechalertpaisarn et al. / Tetrahedron Letters 51 (2010) 5822–5826
5825
4.00
2.00
0.00
-2.00
-4.00
-6.00
-8.00
-10.00
-12.00
(2'R,4'R)-PNA
(2'R,4'R)-PNA+DNA
(2'R,4'S)-PNA
(2'R,4'S)-PNA+DNA
200
220
240
260
280
300
320
Wavelength (nm)
Figure 2. CD spectra of self-complementary (20R,40R)- and (20R,40S)-acpcPNA P3 in the absence and presence of complementary DNA. The spectra were measured at a fixed
concentration of PNA strand = 1 M and DNA strand = 1 M in 10 mM sodium phosphate buffer (pH 7.0) containing 100 mM of NaCl at 20 °C.
l
l
by double duplex invasion,21 the self-pairing may also be useful for
applications including the construction of PNA-based nano-
materials.22
6. Review: Cheng, R. P.; Gellman, S. H.; DeGrado, W. F. Chem. Rev. 2001, 101,
3219–3232.
7. (a) Vilaivan, T.; Suparpprom, C.; Harnyuttanakorn, P.; Lowe, G. Tetrahedron Lett.
2001, 42, 5533–5536; (b) Vilaivan, T.; Lowe, G. J. Am. Chem. Soc. 2002, 124,
9326–9327; (c) Suparpprom, C.; Srisuwannaket, C.; Sangvanich, P.; Vilaivan, T.
Tetrahedron Lett. 2005, 46, 2833–2837; (d) Vilaivan, T.; Srisuwannaket, C. Org.
Lett. 2006, 8, 1897–1900; (e) Vilaivan, T.; Suparpprom, C.; Duanglaor, P.;
Harnyuttanakorn, P.; Lowe, G. Tetrahedron Lett. 2003, 44, 1663–1666.
8. Séquin, U. Experientia 1973, 29, 1059–1062.
9. Morvan, F.; Rayner, B.; Imbach, J. L.; Lee, M.; Hartley, J. A.; Chang, D. K.; Lown, J.
W. Nucleic Acids Res. 1987, 15, 7027–7044.
10. (a) Nielsen, P.; Christensen, N. K.; Dalskov, J. K. Chem. Eur. J. 2002, 8, 712–722;
(b) Laurent, A.; Naval, M.; Debart, F.; Vasseur, J.-J.; Rayner, B. Nucleic Acids Res.
1999, 27, 4151–4159.
Acknowledgements
Financial support from the Thailand Research Fund(RTA5280002
to T.V.), the Development and Promotion of Science and Technology
Talents Project (DPST) (to J.T. and C.B.) and the National Center of
Excellence for Petroleum, Petrochemicals and Advanced Materials
(NCE-PPAM) is acknowledged.
11. Thuong, N. T.; Asseline, U.; Roig, V.; Takasugi, M.; Hélène, C. Proc. Natl. Acad. Sci.
U.S.A. 1987, 84, 5129–5133.
12. Lowe, G.; Vilaivan, T. J. Chem. Soc., Perkin Trans. 1 1997, 547–554.
13. Spectroscopic data of PNA monomers:
Supplementary data
Compound 3a (T monomer): ½a D25
ꢂ
ꢃ2.90 (c 1.0, DMF); 1H NMR (400 MHz,
Supplementary data (experimental details, copies of 1H and 13
C
DMSO-d6) (ratio minor/major isomer ꢀ1:1.38) dH 1.80 (s, 3H, CH3 thymine),
2.20–2.32 and 2.58–2.75 [br m, 2H, CH2(30) rotamers], 3.44–3.49 and 3.73–3.82
[m, 2H, CH2(50) rotamers], 4.18–4.33 (m, 3H, CH Fmoc and CH2 Fmoc), 4.40–
4.43 and 4.53–4.57 [m, 1H, CH(20) rotamers], 5.03–5.11 [m, 1H, CH(40)], 7.31–
7.35 [m, 3H, CH(6) thymine and CH Ar Fmoc], 7.40–7.45 (m, 2H, CH Ar Fmoc),
7.59 and 7.62 (2 ꢄ s, 1H, NH thymine), 7.64–7.66 (m, 2H, CH Ar Fmoc), 7.89–
7.91 (m, 2H, CH Ar Fmoc), 11.32 and 11.35 (2 ꢄ s, 1H, COOH); 13C NMR
(100 MHz, DMSO-d6) dC 12.6 (CH3 thymine), 32.7 and 33.8 [CH2(30) rotamers],
47.0 and 47.1 (CH Fmoc), 49.3 and 49.5 [CH(50) rotamers], 52.3 and 53.3
[CH(40) rotamers], 57.9 and 58.1 [CH(20) rotamers], 67.4 and 67.7 (CH2 Fmoc
rotamers), 110.0 [C(5) thymine], 120.6 (CH Ar Fmoc), 125.6 (CH Ar Fmoc),
127.6 (CH Ar Fmoc), 128.2 (CH Ar Fmoc), 138.1 [C(6)H thymine], 141.1 (C Ar
Fmoc), 144.1 (C Ar Fmoc), 151.4 [C(2) thymine], 154.0 and 154.3 (CO Fmoc
rotamers), 164.2 [C(4) thymine], 173.5 and 173.8 (COOH rotamers); HRMS
(ESI) calcd for C25H23N3O6ꢁH+ 462.1665, found 462.1654.
NMR spectra of compounds 1b, 2a–f and 3a–d, mass spectra of
(20R,40R)-acpcPNA P1–P3 and Tm curves of P2 hybrids) associated
with this article can be found, in the online version, at
doi:10.1016/j.tetlet.2010.08.102.
References and notes
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3. (a) Nielsen, P. E.; Egholm, M.; Berg, R. H.; Buchardt, O. Science 1991, 254, 1497–
1500; (b) Egholm, M.; Buchardt, O.; Nielsen, P. E.; Berg, R. H. J. Am. Chem. Soc.
1992, 114, 1895–1897.
Compound 3b (CBz monomer): ½a 2D4
ꢂ
ꢃ15.1 (c 1.0, DMF); 1H NMR (400 MHz,
DMSO-d6) (ratio minor/major isomer ꢀ1:1.37) dH 2.34–2.47 and 2.71–2.86 [br
m, 2H, CH2(30) rotamers], 3.59–3.63 and 3.85–3.94 [m, 2H, CH2(50) rotamers],
4.19–4.38 (m, 3H, CH2 and CH Fmoc rotamers), 4.49 (dd J = 8.8, 3.3 Hz) and
4.63 (m) [1H, CH(20) rotamers], 5.14 [m, 1H, CH(40)], 7.30–7.38 [m, 3H, CH Ar
Fmoc and CH(5) cytosine], 7.33–7.45 (m, 2H, CH Ar Fmoc), 7.51–7.55 (m, 2H,
CH Ar Fmoc), 7.62–7.69 (m, 3H, CH Bz), 7.88–7.91 (m, 2H, CH Ar Fmoc), 8.00–
8.05 (m, 2H, CH Bz), 8.14 (d J = 7.5 Hz) and 8.19 (d J = 7.5 Hz) [1H, CH(6)
cytosine rotamers], 11.28 (br s, 1H, COOH); 13C NMR (100 MHz, DMSO-d6) dC
32.9 and 34.1 [CH2(30) rotamers], 47.0 and 47.1 (CH Fmoc rotamers), 50.1 and
50.5 [CH2(50) rotamers], 55.4 and 56.4 [CH(40) rotamers], 57.9 and 58.1 [CH(20)
rotamers], 67.3 and 67.7 (CH2 Fmoc rotamers), 96.9 [C(5)H cytosine], 120.6 (CH
Ar Fmoc), 125.6 (CH Ar Fmoc), 127.6 (CH Ar Fmoc), 128.2 (CH Ar Fmoc), 128.9
4. (a) Kumar, V. A. Eur. J. Org. Chem. 2002, 2021–2032; (b) Kumar, V. A.; Ganesh, K.
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Andrasi, A.; Rapireddy, S.; Frezza, B. M.; Gayathri, C.; Gil, R. R.; Ly, D. H. J. Am.
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Biomol. Chem. 2008, 6, 92–103.

5826
J. Taechalertpaisarn et al. / Tetrahedron Letters 51 (2010) 5822–5826
(CH Bz), 133.2 (CH Bz), 133.6 (C Bz), 141.1 (C Ar Fmoc), 144.1 (C Ar Fmoc),
rotamers], 57.8 and 58.1 [CH(20) rotamers], 67.3 and 67.7 (CH2 Fmoc rotamers),
120.6 (CH Ar Fmoc), 120.7 [C(5)H guanine], 125.5 and 125.7 (CH Ar Fmoc
rotamers), 127.5 and 127.6 (CH Ar Fmoc rotamers), 128.1 and 128.2 (CH Ar
Fmoc rotamers), 138.3 [C(8)H guanine], 141.2 (C Ar Fmoc), 144.1 (C Ar Fmoc),
148.4 and 149.0/149.1 [C(6) and C(2) guanine], 154.0 and 154.2 (CO Fmoc
rotamers), 155.3 [C(4) guanine], 173.4 and 173.7 (COOH rotamers), 180.7 (CO
Ibu); HRMS (ESI+) calcd for C29H28N6O6ꢁH+ 557.2149, found 557.2100.
14. LePlae, P. R.; Umezawa, N.; Lee, H. S.; Gellman, S. H. J. Org. Chem. 2001, 66,
5629–5632.
15. The pentafluorophenyl esters were generated from the corresponding Fmoc
free acids by treatment with pentafluorophenyl trifluoroacetate/DIEA. The
unstable active esters were used following an acid-base extraction to remove
DIEA salts without further purification.
16. Lowe, G.; Vilaivan, T. J. Chem. Soc., Perkin Trans. 1 1997, 555–560.
17. In contrast to homopyrimidine aegPNA which readily forms (PNA)2ꢁDNA
hybrids, it was established from our previous titration experiments (Refs. 7a–
d) that homothymine pyrrolidinyl PNA forms only 1:1 hybrids with DNA.
18. Heating of single-stranded (20R,40S)-acpcPNA with a non-self-complementary
sequence such as P1 resulted in no melting curve. This rules out the
possibilities of intramolecular folding or non-specific aggregate formation
such as those observed in aegPNA.
19. (a) Kitamatsu, M.; Shigeyasu, M.; Okada, T.; Sisido, M. Chem. Commun. 2004,
1208–1209; (b) Kitamatsu, M.; Shigeyasu, M.; Saitoh, M.; Sisido, M.
Biopolymers 2006, 84, 267–273; (c) Lonkar, P. S.; Ganesh, K. N.; Kumar, V. A.
Org. Biomol. Chem. 2004, 2, 2604–2611.
147.6 [C(6)H cytosine], 154.1 and 154.2 (CO Fmoc rotamers), 155.4 [C(2)
cytosine], 163.2 [C(4) cytosine], 168.0 (CO Bz), 173.5 and 173.8 (COOH
rotamers); HRMS (ESI+) calcd for C31H26N4O6ꢁH+ 551.1931, found 551.1873.
Compound 3c (ABz monomer): ½a 2D4
ꢂ
+7.70 (c 1.0, DMF); 1H NMR (400 MHz,
DMSO-d6) (ratio minor/major isomer ꢀ1:1.36) dH 2.54–2.68 and 3.01–3.18 [m,
2H, CH2(30) rotamers], 3.87–3.98 and 4.03–4.07 [m, 2H, CH2(50) rotamers],
4.20–4.38 (m, 3H, CH2 Fmoc and CH Fmoc rotamers), 4.60 (dd J = 8.9, 3.8 Hz)
and 4.76 (dd J = 9.0, 3.1 Hz) [1H, CH(20) rotamers], 5.37 [m, 1H, CH(40)], 7.21–
7.45 (m, 4H, CH Ar Fmoc), 7.55–7.70 (m, 5H, CH Ar Fmoc and Bz), 7.82–7.91 (m,
2H, CH Ar Fmoc), 8.06 (m, 2H, CH Bz), 8.59 and 8.62 [2 ꢄ s, 1H, CH(8) adenine
rotamers], 8.75 and 8.78 [2 ꢄ s, 1H, CH(2) adenine rotamers], 11.22 (br s, 1H,
COOH); 13C NMR (100 MHz, DMSO-d6) dC 33.5 and 34.7 [CH2(30) rotamers],
46.5 and 46.6 (CH Fmoc rotamers), 50.2 and 50.6 [CH2(50) rotamers], 52.2 and
53.0 [CH(40) rotamers], 57.5 and 57.7 [CH(20) rotamers], 66.9 and 67.3 (CH2
Fmoc rotamers), 120.1 (CH Ar Fmoc), 125.0 and 125.2 (CH Ar Fmoc), 125.6
[C(5) adenine], 127.1 and 127.7 (CH Ar Fmoc), 128.5 (CH Bz), 132.4 (CH Bz),
133.4 (C Bz), 140.6 (C Ar Fmoc), 143.3 and 143.4 [C(8)H adenine rotamers],
143.6 (C Ar Fmoc), 150.3 [C(4) adenine], 151.3 [C(2)H adenine], 152.2 [C(6)
adenine], 153.8 (CO Fmoc), 165.8 (CO Bz), 173.0 and 173.3 (COOH rotamers);
HRMS (ESI+) calcd for C32H26N6O5ꢁH+ 575.2043, found 575.2095.
Compound 3d (GIbu monomer): ½a 2D3
ꢂ
ꢃ3.43 (c 0.5, DMF); 1H NMR (400 MHz,
DMSO-d6) (ratio minor/major isomer ꢀ1:1.32) dH 1.13 (d, J = 6.0 Hz) and 1.14
(d, J = 6.5 Hz) (6H, CH3 Ibu rotamers), 2.45–2.61 and 2.89–3.04 [m, 2H, CH2(30)
rotamers], 2.78 (m, 1H, CH Ibu), 3.69–3.78 and 3.94–4.02 [m, 2H, CH2(50)
rotamers], 4.19–4.38 (m, 3H, CH2 Fmoc and CH Fmoc rotamers), 4.51 (dd
J = 8.7, 2.9 Hz) and 4.65 (dd J = 8.8, 2.2 Hz) [1H, CH(20) rotamers], 5.06–5.14 [m,
1H, CH(40)], 7.23–7.45 (m, 4H, CH Ar Fmoc rotamers), 7.59–7.68 (m, 2H, CH Ar
Fmoc rotamers), 7.84–7.91 (m, 2H, CH Ar Fmoc rotamers), 8.14 and 8.18 [2 ꢄ s,
1H, CH(8) guanine rotamers], 11.73 and 11.76 (2 ꢄ s, 1H, NH/OH rotamers),
12.10 and 12.12 (2 ꢄ s, 1H, NH/OH rotamers); 13C NMR (100 MHz, DMSO-d6) dC
19.3 (CH3 Ibu), 34.1 and 35.3 [CH2(30) rotamers], 35.2 (CH Ibu), 47.0 and 47.1
(CH Fmoc rotamers), 50.9 and 51.3 [CH2(50) rotamers], 51.7 and 52.7 [CH(40)
20. (a) Boontha, B.; Nakkuntod, J.; Hirankarn, N.; Chaumpluk, P.; Vilaivan, T. Anal.
Chem. 2008, 80, 8178–8186; (b) Ananthanawat, C.; Vilaivan, T.; Hoven, V. P.;
Su, X. Biosens. Bioelectron. 2010, 25, 1064–1069.
21. (a) Lohse, J.; Dahl, O.; Nielsen, P. E. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 11804–
11808; (b) Ishizuka, T.; Yoshida, J.; Yamamoto, Y.; Sumaoka, J.; Tedeschi, T.;
Corradini, R.; Sforza, S.; Komiyama, M. Nucleic Acids Res. 2008, 36, 1464–1471.
22. (a) Gothelf, K. V.; LaBean, T. H. Org. Biomol. Chem. 2005, 3, 4023–4037; (b) Ito,
Y.; Fukusaki, E. J. Mol. Catal. B: Enzym. 2004, 128, 155–166.