(S,S)-trans-cyclopentane-constrained peptide nucleic acids. A general backbone modification that improves binding affinity and sequence specificity

Article abstract of DOI:10.1021/ja046280q

Replacing the ethylenediamine portion of aminoethylglycine peptide nucleic acids (aegPNAs) with one or more (S,S)-trans-cyclopentane diamine units significantly increases binding affinity and sequence specificity to complementary DNA, making these modifie

Full text of DOI:10.1021/ja046280q

Published on Web 11/02/2004
(S,S)-trans-Cyclopentane-Constrained Peptide Nucleic Acids.
A General Backbone Modification that Improves Binding
Affinity and Sequence Specificity
Jonathan K. Pokorski, Mark A. Witschi, Bethany L. Purnell, and Daniel H. Appella*
Contribution from the Department of Chemistry, Northwestern UniVersity,
EVanston, Illinois 60208
Received June 23, 2004; E-mail: dappella@chem.northwestern.edu
Abstract: Replacing the ethylenediamine portion of aminoethylglycine peptide nucleic acids (aegPNAs)
with one or more (S,S)-trans-cyclopentane diamine units significantly increases binding affinity and sequence
specificity to complementary DNA, making these modified PNAs ideal for use as nucleic acid probes in
genomic analysis. The synthesis and study of this new class of PNAs (tcypPNAs) is described in which
trans-cyclopentane diamine has been incorporated into several positions, and in varying number, within
PNA backbones of mixed-base sequences.
Introduction
sequences with significantly higher affinity than the correspond-
ing DNA sequences,7 which improves sensitivity. PNA is also
In this paper, we report that incorporating one or more (S,S)-
trans-cyclopentane diamine units into aminoethylglycine peptide
nucleic acids (aegPNAs)1 significantly increases binding affinity
and sequence specificity to complementary DNA. With the
completion of the human genome sequence, and the sequences
of several other organisms, genomic analysis can provide key
diagnostic information about diseases and pathogens, as well
as impact biochemical and biomedical research.2 Central to the
success of such studies is the availability of reliable diagnostic
techniques to signal the presence or absence of specific DNA
sequences. Many recent technological advances have shown that
synthetic DNA oligomers can be coupled to highly sensitive
assays so that the binding of a complementary DNA sequence
can be detected.3 These devices are typically rated according
to their ability to detect low levels of DNA, discriminate between
very similar sequences of DNA (i.e., identify single nucleotide
polymorphisms (SNPs)), and provide useful information in a
reasonable time frame. In particular, DNA microarrays are now
routinely used to quantify gene expression and identify SNPs
in a wide variety of biological studies;4 however, limitations of
the technology still exist.5
significantly more stable on surfaces than DNA, yielding
detection devices with longer shelf lives.8 Most importantly,
PNA-based probes can bind to complementary DNA at low salt
concentrations in solution.9 To form DNA-DNA duplexes, a
high concentration of positively charged counterions is required
to neutralize the charge-charge repulsion associated with
bringing together two anionic backbones.10 In addition, high
salt concentration can promote formation of structures in single-
stranded DNA via intramolecular hydrogen bonding, and
promote formation of tertiary structures.10d Since PNAs consist
of a neutral polyamide backbone, counterions are not required
for the formation of PNA-DNA duplexes. By destabilizing
DNA structures under low salt concentrations, one can create
conditions that favor formation of DNA-PNA duplexes over
DNA-DNA duplexes and tertiary structures. This arrangement
should give PNA-based detection probes a distinct advantage
over DNA probes because the chance for false negatives should
be reduced. Despite the benefits that PNA can offer to DNA
diagnostics, the use of PNA probes lags far behind that of DNA
probes, most likely because the most commonly employed PNAs
(derived from an aminoethylglycine backbone) do not have
significantly improved properties over DNA to warrant the effort
or funds necessary to purchase, license, or synthesize these
oligomers.11
Replacing DNA probes with PNA has been shown to improve
DNA-detection technology.6 PNAs bind to complementary DNA
(1) (a) Nielsen, P. E.; Egholm, M.; Berg, R. H.; Buchardt, O. Science 1991,
254, 1497. (b) Nielsen, P. E. Curr. Opin. Biotechnol. 2001, 12, 16. (c)
Nielsen, P. E. Mol. Biotechnol. 2004, 26, 233.
(2) Lockhart, D. J.; Winzeler, E. A. Nature 2000, 405, 827.
(3) (a) Meldrum, D. Genome Res. 2000, 10, 1288. (b) Taton, T. A.; Mirkin,
C. A.; Letsinger, R. L. Science 2000, 289, 1757. (c) Vainrub, A.; Pettitt,
B. M. J. Am. Chem. Soc. 2003, 125, 7798. (d) Ivnitski, D.; O’Neil, D. J.;
Gattuso, A.; Schlicht, R.; Calidonna, M.; Fisher, R. Biotechniques 2003,
35, 862.
(7) Ratilainen, T.; Holme´n, A.; Tuite, E.; Nielsen, P. E.; Norde´n, B.
Biochemistry 2000, 39, 7781.
(8) Kroger, K.; Jung, A.; Reder, S.; Gauglitz, G. Anal. Chim. Acta 2002, 469,
37.
(9) Weiler, J.; Gausepohl, H.; Hauser, N.; Jensen, O. N.; Hoheisel, J. D. Nucleic
Acids Res. 1997, 25, 2792.
(4) (a) Haviv, I.; Campbell, I. G. Mol. Cell. Endocrinol. 2002, 191, 121. (b)
Bustin, S. A.; Dorudi, S. Trends Mol. Med. 2002, 8, 269.
(5) Tan, P. K.; Downey, T. J.; Spitznagel, E. L.; Xu, P.; Fu, D.; Dimitrov, D.
S.; Lempicki, R. A.; Raaka, B. M.; Cam, M. C. Nucleic Acids Res. 2003,
31, 5676.
(10) (a) Marmur, J.; Doty, P. J. Mol. Biol. 1962, 5, 109. (b) Schildkraut, C.;
Lifson, S. Biopolymers 1965, 3, 195. (c) Braunlin, W. H.; Bloomfield, V.
A. Biochemistry 1991, 30, 754. (d) Bowater, R. P.; Aboul-ela, F.; Lilley,
D. M. J. Nucleic Acids Res. 1994, 22, 2042. (e) Anderson, C. F.; Record,
M. T. Annu. ReV. Phys. Chem. 1995, 46, 657.
(11) Chandler, D. P.; Stults, J. R.; Anderson, K. K.; Cebula, S.; Schuck, B. L.;
Brockman, F. J. Anal. Biochem. 2000, 283, 241.
(6) (a) Igloi, G. L. Expert ReV. Mol. Diagn. 2003, 3, 17. (b) Demidov, V. V.;
Frank-Kamenetskii, M. D. Trends Biochem. Sci. 2004, 29, 62.
9
10.1021/ja046280q CCC: $27.50 © 2004 American Chemical Society
J. AM. CHEM. SOC. 2004, 126, 15067-15073
15067

A R T I C L E S
Pokorski et al.
Figure 1. General strategy to insert trans-cyclopentane into the aegPNA backbone.
([R]D) were measured on a Perkin-Elmer 241 polarimeter using sodium
light (D line, 589.3 nm) and are reported in degrees; concentrations
(c) are reported as g/100 mL. Infrared spectra (IR) were obtained on a
Bio-Rad FTS60 FTIR. IR spectra were collected either as a thin film
on a KBr disk or by preparing a pressed KBr pellet containing the title
compound. Proton nuclear magnetic resonances (1H NMR) were
recorded in deuterated solvents on an iNOVA 500 (500 MHz)
spectrometer. Chemical shifts are reported in parts per million (ppm,
δ) relative to tetramethylsilane (δ 0.00). If tetramethylsilane was not
present, the residual protio solvent is referenced (CDCl3, δ 7.27;
dimethyl sulfoxide-d6 (DMSO), δ 2.50). 1H NMR splitting patterns are
designated as singlet (s), doublet (d), or quartet (q). Splitting patterns
that could not be interpreted or easily visualized were designated as
multiplet (m) or broad (br). Coupling constants are reported in hertz
(Hz). Proton-decoupled (13C NMR) spectra were obtained on an iNOVA
500 (125 MHz) spectrometer and are reported in ppm using the solvent
as an internal standard (CDCl3, δ 77.23; DMSO, δ 39.52). Low-
resolution mass spectra (LRMS) were obtained using a Micromass
Quattro II triple quadrupole HPLC/MS/MS mass spectrometer. High-
resolution mass spectra (HRMS) were obtained on a Micromass Q-Tof
Ultima at the University of Illinois at Urbana-Champaign Mass
Spectrometry Center. Elemental analysis data were collected by Atlantic
Microlab, Inc. Analytical thin-layer chromatography (TLC) was carried
out on Sorbent Technologies TLC plates precoated with silica gel (250
µm layer thickness). Flash column chromatography was performed on
EM Science silica gel 60 (230-400 mesh). Solvent mixtures used for
TLC and flash column chromatography are reported in v/v ratios. Unless
otherwise noted, all commercially available reagents and solvents were
purchased from Aldrich and used without further purification. Tetra-
hydrofuran (THF) was distilled from sodium and benzophenone prior
to use. Dimethylformamide (DMF) was purified by passage through a
bed of activated alumina.16 1-(3-Dimethylaminopropyl)-3-ethylcarbo-
diimide hydrochloride (EDC), HATU, and Boc-Lys-(2-Cl-Z)-OH were
purchased from Advanced ChemTech. All aegPNA monomers were
purchased from Applied Biosystems. Kaiser test solutions were
purchased from Fluka.
In this paper, we describe the synthesis and study of a new
class of trans-cyclopentane-derived PNAs (tcypPNAs) in which
trans-cyclopentane diamine has been incorporated into several
positions, and in varying number, within PNA backbones of
mixed-base sequences (Figure 1). Our initial publication on this
research described a first-generation synthesis and initial binding
studies of these modified PNAs.12 We now report a more
efficient, second-generation synthesis of tcypPNAs, and present
binding data demonstrating the improvements that trans-
cyclopentane incorporation conveys to PNAs. Compared to
unmodified PNA, tcypPNA displays improved binding affinity
(i.e., higher Tm’s) and sequence specificity to complementary
DNA, indicating that these modified PNAs possess favorable
properties for use in DNA diagnostics. Furthermore, introduction
of cyclopentanes into the PNA backbone affords PNAs with
general and additive improvements in binding affinity to
complementary DNA. While several other PNA backbone
modifications have been explored, most lead to reduction in
the stability of a PNA-DNA duplex.13 Among the few modified
PNAs that afford higher PNA-DNA stability, most are limited
for one or more of the following reasons: the modified residues
are synthetically challenging, the modification does not increase
the stability of the duplex in a general manner, or cationic groups
must be employed in order to promote binding affinity (which
could ultimately lead to nonspecific binding to other DNA
sequences or anionic biomolecules).14,15 While our work was
in progress, Kumar and Ganesh reported that cis-cyclopentane
in oligothymine PNAs greatly stabilizes PNA2DNA triplexes.14e,i
The trans-cyclopentane modification described in our paper
increases the stability and sequence specificity of PNA-DNA
duplexes composed of mixed-base sequences, and provides a
new class of probes that can be coupled to analytical devices
for DNA detection.
Abbreviations: RBF, round-bottom flask; rt, room temperature;
DMAP, dimethylaminopyridine; DCM, dichloromethane; DIEA, N,N-
diisopropylethylamine; NMP, 1-methyl-2-pyrrolidinone; HATU, O-(7-
azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophos-
phate; MDCHA, N-methyldicyclohexylamine; HBTU, O-(benzotriazol-
1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate; Ac2O, acetic
anhydride; TFMSA, trifluoromethanesulfonic acid; TFA, trifluoroacetic
acid.
(1S,2S)-2-(tert-Butoxycarbonylamino)cyclopentyl Isocyanate (2).
â-Amino acid 117 (2.3 g, 10.1 mmol) was added to an oven-dried 250
mL RBF under N2(g) atmosphere and dissolved in dry THF (50 mL)
with stirring. The solution was cooled to 0 °C, and triethylamine (2.8
mL, 20.2 mmol, 2.0 equiv) and ethyl chloroformate (1.9 mL, 20.2
Experimental Section
General. Melting points (mp) were obtained on a Thomas-Hoover
capillary melting point apparatus and are uncorrected. Optical rotations
(12) Myers, M. C.; Witschi, M. A.; Larionova, N. V.; Franck, J. M.; Haynes,
R. D.; Hara, T.; Grajkowski, A.; Appella, D. H. Org. Lett. 2003, 5, 2695.
(13) Ganesh, K. N.; Nielsen, P. E. Curr. Org. Chem. 2000, 4, 931.
(14) For examples of other modified PNAs that bind DNA, see: (a) Kumar, V.
A. Eur. J. Org. Chem. 2002, 2021 and references therein. (b) Govindaraju,
T.; Gonnade, R. G.; Bhadbhade, M. M.; Kumar, V. A.; Ganesh, K. N.
Org. Lett. 2003, 5, 3013. (c) Hollenstein, M.; Leumann, C. J. Org. Lett.
2003, 5, 1987. (d) Sharma, N. K.; Ganesh, K. N. Chem. Commun. 2003,
2484. (e) Govindaraju, T.; Kumar, V. A.; Ganesh, K. N. Chem. Commun.
2004, 860. (f) Govindaraju, T.; Kumar, V. A.; Ganesh, K. N. J. Org. Chem.
2004, 69, 1858. (g) Lonkar, P. S.; Kumar, V. A. Bioorg. Med. Chem. Lett.
2004, 14, 2147. (h) Olsen, A. G.; Dahl, O.; Nielsen, P. E. Bioorg. Med.
Chem. Lett. 2004, 14, 1551. (i) Govindaraju, T.; Kumar, V. A.; Ganesh,
K. N. J. Org. Chem. 2004, 69, 5725.
(16) Pangborn, A. B.; Giardello, M. A.; Grubbs, R. H.; Rosen, R. K.; Timmers,
F. J. Organometallics 1996, 15, 1518.
(17) LePlae, P. R.; Umezawa, N.; Lee, H. S.; Gellman, S. H. J. Org. Chem.
2001, 66, 5629.
(15) For a discussion on nonspecific binding of cationic PNAs to DNA, see:
Abibi, A.; Protozanova, E.; Demidov, V. V.; Frank-Kamenetskii, M. D.
Biophys. J. 2004, 86, 3070.
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(S,S)-trans-Cyclopentane-Constrained Peptide Nucleic Acids
A R T I C L E S
mmol, 2.0 equiv) were added dropwise via syringe, forming a white
precipitate. The heterogeneous mixture was stirred at 0 °C for 20 min.
Sodium azide (2.0 g, 30.3 mmol, 3.0 equiv) was dissolved in H2O (35
mL) and added to the reaction mixture. The biphasic mixture was stirred
vigorously at 0 °C for 20 min. The ice bath was removed, and the
mixture was allowed to warm to room temperature for 10 min. The
mixture was diluted with H2O (30 mL) and was extracted with ethyl
acetate (3 × 30 mL). The combined organic phases were dried over
Na2SO4 and concentrated (not to dryness!).18 The residue was taken
up in benzene (50 mL) and refluxed for 30 min. The solution was
cooled, concentrated, and dried under vacuum to yield 2.15 g (94%)
of 2 as a colorless, crystalline solid: mp ) 73-75 °C; IR (KBr) 3700,
3379, 2981, 2279 (NdCdO stretch), 1687, 1517, 1458, 1366, 1345,
crystalline solid upon sitting at room temperature: Rf ) 0.30 (EtOAc);
mp ) 49-52 °C; [R]23 ) +5.0° (c ) 1.0, EtOH, 100%); IR (film)
D
3343, 2960, 2871, 1741, 1703, 1522, 1438, 1392, 1367, 1244, 1207,
1173, 1044, 1021, 996, 778 cm-1; 1H NMR (500 MHz, CDCl3) δ 4.56
(br s, 1H, Boc-NH), 3.73 (s, 3H, CO2CH3), 3.67 (br m, 1H, Boc-
NH-CH), 3.48 (q, J ) 13.5 Hz, 2H, NH-CH2-CO2Me), 2.85 (m,
1H, CH-NH), 2.13 (m, 1H, cyclopentane-H), 1.93 (m, 2H, cyclo-
pentane-H), 1.77-1.68 (m, 1H, cyclopentane-H), 1.67-1.59 (m, 1H,
cyclopentane-H), 1.45 (s, 9H, tert-butyl-CH3), 1.42-1.34 (m, 1H,
cyclopentane-H); 13C NMR (125 MHz, CDCl3) δ 172.8, 155.5, 78.6,
64.7, 57.2, 51.4, 48.8, 31.1, 30.8, 28.1, 21.2; LRMS (ESI-MS m/z)
mass calcd for C13H25N2O4 [M + H]+, 273.18, found 273.2. Anal. Calcd
for C13H24N2O4: C, 57.33; H, 8.88; N, 10.29. Found: C, 57.20; H,
8.96; N, 10.22.
1
1314, 1290, 1250, 1164, 1047, 909, 780, 580 cm-1; H NMR (500
MHz, CDCl3) δ 4.48 (br s, 1H, Boc-NH), 3.85 (br s, 1H, OdCdNs
CH), 3.71 (br s, 1H, Boc-NH-CH), 2.19-2.11 (m, 1H, cyclopentane-
H), 2.04-1.97 (m, 1H, cyclopentane-H), 1.81-1.65 (m, 3H, cyclo-
pentane-H), 1.46 (s, 10H, tert-butyl-CH3 + cyclopentane-H).
General Procedure A for Coupling Bases to tcypPNA Backbone.
Compound 4 (0.9 g, 3.5 mmol) was added to an oven-dried 50 mL
RBF and dissolved in dry DMF (15 mL) with stirring. The solution
was cooled to 0 °C. Nucleobase acetic acid (5.25 mmol, 1.5 equiv)
and DMAP (100 mg, 0.9 mmol) were added. EDC (1.3 g, 7.0 mmol,
2.0 equiv) was added, and the reaction mixture was allowed to stir at
0 °C for 10 min. The ice bath was removed, and the reaction was stirred
for 36 h at room temperature. The solution was diluted with H2O (90
mL) and extracted with ethyl acetate (3 × 60 mL). The combined
organic phases were washed with saturated aqueous NaCl (4 × 75 mL),
dried over Na2SO4, concentrated, and dried under vacuum to yield base-
coupled monomer esters as colorless solids.
Methyl N-[(2S)-tert-Butoxycarbonylaminocyclopent-(1S)-yl]-N-
[thymin-1-ylacetyl]glycinate (5). Following general procedure A,
tcypPNA backbone 4 (0.95 g, 3.5 mmol) was coupled to thymine acetic
acid (0.96 g, 5.25 mmol) to yield 1.33 g (87%) of ester 5. If necessary,
the solid was purified by flash column chromatography: Rf ) 0.38 (5%
MeOH/CH2Cl2); 1H NMR (500 MHz, DMSO) δ major rotamer 11.29
(s, 1H, imide-NH), 7.17 (s, 1H, thymine-H), 6.96 (d, J ) 8.0 Hz,
1H, Boc-NH), 4.72 (q, J ) 16.5 Hz, 2H, thymine-CH2), 4.0-3.75
(m, 4H, NH-CH2-CO2Me + CH-NH), 3.59 (s, 3H, CO2CH3), 1.9-
1.4 (m, 6H, cyclopentane-H), 1.75 (s, 3H, thymine-CH3), 1.36 (s,
9H, tert-butyl-CH3), minor rotamer 7.20 (s, 1H, thymine-H), 6.75
(d, J ) 8.0 Hz, 1H, Boc-NH), 4.45 (q, J ) 16.5 Hz, 2H, thymine-
CH2), 3.70 (s, 3H, CO2CH3); 13C NMR (125 MHz, DMSO) δ 169.6,
167.3, 164.4, 155.4, 151.0, 141.7, 108.3, 77.9, 61.1, 52.7, 51.6, 47.9,
43.6, 28.2, 25.9, 18.8, 12.0; LRMS (ESI-MS m/z) mass calcd for
C20H31N4O7 [M + H]+, 439.22, found 439.2.
tert-Butyl
(1S,2S)-2-(Benzyloxycarbonylamino)cyclopentyl-
carbamate (3).19 Copper(I) chloride (0.9 g, 9.5 mmol) was added to
an oven-dried 100 mL RBF under N2(g) atmosphere and suspended in
dry DMF (35 mL) with stirring. Benzyl alcohol (0.9 mL, 9.5 mmol)
was added dropwise via syringe and allowed to stir until a bright yellow-
green suspension had formed (approximately 5 min). At this point, 2
(2.1 g, 9.5 mmol) was added and stirred at room temperature for 45
min. The dark green suspension was poured into H2O (70 mL) and
extracted with ethyl acetate (150 mL). The organic phase was washed
with saturated aqueous NaCl (2 × 50 mL), dried over Na2SO4,
concentrated, and dried under vacuum to yield 2.83 g (89%) of 3 as a
colorless solid: Rf ) 0.38 (2% MeOH/ CH2Cl2); mp ) 150-151 °C;
[R]23 ) -8.0° (c ) 1.0, EtOH, 100%); IR (film) 3341, 2971, 1680,
D
1528, 1304, 1270, 1233, 1169, 1041, 779, 741, 696, 639 cm-1; 1H NMR
(500 MHz, CDCl3) δ 7.34 (m, 5H, Ph-H), 5.30 (br s, 1H, Cbz-NH),
5.09 (s, 2H, Ph-CH2), 4.81 (br s, 1H, Boc-NH), 3.68 (m, 2H,
carbamate-NH-CH), 2.17-2.09 (m, 2H, cyclopentane-H), 1.70 (m,
2H, cyclopentane-H), 1.43 (s, 11H, tert-butyl-CH3 + 2 cyclopentane-
H); 13C NMR (125 MHz, CDCl3) δ 156.9, 156.5, 136.7, 128.5, 128.0,
79.5, 66.6, 58.4, 57.2, 30.1, 29.8, 28.4, 19.7; LRMS (ESI-MS m/z):
mass calcd for C18H27N2O4 [M + H]+, 335.20, found 335.3. Anal. Calcd
for C18H26N2O4: C, 64.65; H, 7.84; N, 8.38. Found: C, 64.45; H, 7.86;
N, 8.29.
tert-Butyl
(1R,2R)-2-(Benzyloxycarbonylamino)cyclopentyl-
Methyl N-[(2S)-tert-Butoxycarbonylaminocyclopent-(1S)-yl]-N-
[4-N-(benzyloxycarbonyl)cytosin-1-ylacetyl]glycinate (6). Following
general proedure A, tcypPNA backbone 4 (0.84 g, 2.94 mmol) was
coupled to Cbz-cytosine acetic acid20 (1.42 g, 4.70 mmol) to yield 0.88
g (52%) of ester 6. If necessary, the solid was purified by flash column
chromatography: Rf ) 0.21 (1% MeOH/EtOAc); 1H NMR (500 MHz,
DMSO) δ major rotamer 10.77 (s, 1H, Cbz-NH), 7.78 (d, J ) 7.0
Hz, 1H, cytosine-H), 7.41 (m, 5H, Ph-H), 7.01 (d, J ) 7.0 Hz, 1H,
cytosine-H), 6.95 (d, J ) 8.5 Hz, 1H, Boc-NH), 5.19 (s, 2H, Ph-
CH2), 4.90 (q, J ) 16.0 Hz, 2H, cytosine-CH2), 4.35-3.75 (m, 4H,
NH-CH2-CO2Me + CH-NH), 3.58 (s, 3H, CO2CH3), 1.90-1.40 (m,
6H, cyclopentane-H), 1.36 (s, 9H, tert-butyl-CH3), minor rotamer
7.84 (d, J ) 7.0 Hz, 1H, cytosine-H), 6.73 (d, J ) 8.5 Hz, 1H, Boc-
NH), 4.61 (q, J ) 16.0 Hz, 2H, cytosine-CH2), 3.70 (s, 3H, CO2CH3);
13C NMR (125 MHz, CDCl3) δ 169.5, 167.3, 163.0, 155.4, 155.0, 153.2,
150.5, 136.0, 128.5, 128.2, 127.9, 93.9, 77.9, 66.5, 61.1, 52.7, 51.6,
49.5, 43.7, 28.2, 26.0, 18.8; LRMS (ESI-MS m/z) mass calcd for
C27H36N5O8 [M + H]+, 558.26, found 558.1.
carbamate. Prepared via identical procedures used to prepare 3, except
with (R)-R-methylbenzylamine. All characterization data matched 3
except [R]23 ) +8.0° (c ) 1.0, EtOH, 100%).
D
tert-Butyl
(1S,2S)-2-((Methoxycarbonyl)methylamino)cyclo-
pentylcarbamate (4). Compound 3 (1.5 g, 7.6 mmol) was dissolved
in methanol (150 mL) and added to an oven-dried Parr flask containing
10% Pd/C (0.5 g). The flask was placed on a Parr apparatus under 55
psi of H2(g) pressure, and was shaken for 12 h. TLC analysis revealed
no starting material [Rf ) 0.38 (2% MeOH/CH2Cl2)]. The suspension
was filtered through Celite and concentrated. The residue was dissolved
in dry DMF with stirring. Triethylamine (1.1 mL, 7.6 mmol) and methyl
bromoacetate (0.6 mL, 6.8 mmol, 0.9 equiv) were added dropwise via
syringe. The reaction was allowed to stir at room temperature for 3 h.
The reaction mixture was diluted with saturated aqueous NaHCO3 (50
mL) and extracted with ethyl acetate (2 × 35 mL). The combined
organic phases were washed with saturated aqueous NaCl (2 × 25 mL),
dried over Na2SO4, concentrated, and dried under vacuum. The residue
was purified by flash column chromatography [Rf ) 0.30 (EtOAc)] to
yield 1.26 g (67%) of 4 as a colorless oil, which formed a colorless,
Methyl N-[(2S)-tert-Butoxycarbonylaminocyclopent-(1S)-yl]-N-
[6-N-(benzyloxycarbonyl)adenin-9-ylacetyl]glycinate (7). Following
general procedure A, tcypPNA backbone 4 (0.20 g, 0.70 mmol) was
(18) We have performed this reaction over 50 times, typically on 2 g scale, and
we have never had an explosion; however, cyclopentyl acyl-azides have
been reported to explode when concentrated to dryness. See: Ongeri, S.;
Aitken, D. J.; Husson, H. P. Synth. Commun. 2000, 30, 2593.
(20) Thompson, S. A.; Josey, J. A.; Cadilla, R.; Gaul, M. D.; Hassman, F.;
Luzzio, M. J.; Pipe, A. J.; Reed, K. L.; Ricca, D. J.; Wiethe, R. W.; Noble,
S. A. Tetrahedron 1995, 51, 6179.
(19) Duggan, M. E.; Imagire, J. S. Synthesis 1989, 131.
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A R T I C L E S
Pokorski et al.
observedb
coupled to Cbz-adenine acetic acid20 (0.34 g, 1.05 mmol) to yield 0.26
g (63%) of ester 7. If necessary, the solid was purified by flash column
chromatography: Rf ) 0.26 (5% MeOH/CH2Cl2); 1H NMR (500 MHz,
DMSO) δ major rotamer 10.68 (s, 1H, Cbz-NH), 8.59 (s, 1H,
adenine-H), 8.25 (s, 1H, adenine-H), 7.46 (d, J ) 7.5 Hz, 2H, o-Ph-
H), 7.40 (t, J ) 7.5 Hz, 2H, m-Ph-H), 7.34 (t, J ) 7.5 Hz, 1H, p-Ph-
H), 7.08 (br d, J ) 8.5 Hz, 1H, Boc-NH), 5.41 (q, J ) 17.0 Hz, 2H,
adenine-CH2), 5.22 (s, 2H, Ph-CH2), 4.43-3.81 (m, 4H, NH-CH2-
CO2Me + CH-NH), 3.56 (s, 3H, CO2CH3), 1.97-1.46 (m, 6H,
cyclopentane-H), 1.39 (s, 9H, tert-butyl-CH3), minor rotamer 8.29
(s, 1H, adenine-H), 6.75 (d, J ) 8.5 Hz, 1H, Boc-NH), 3.76 (s, 3H,
CO2CH3), 1.36 (s, 9H, tert-butyl-CH3); 13C NMR (125 MHz, DMSO)
δ 169.5, 166.6, 155.4, 152.4, 151.3, 149.1, 145.2, 136.3, 128.4, 128.0,
127.9, 122.0, 78.0, 66.5, 61.3, 52.8, 51.6, 44.3, 43.7, 28.2, 25.9, 18.8;
LRMS (ESI-MS m/z) mass calcd for C28H36N7O7 [M + H]+, 582.27,
found 582.1.
General Procedure B for Saponification of Methyl Esters. A
tcypPNA ester (0.9 g, 2.1 mmol) was dissolved in THF (33 mL) and
cooled to 0 °C. Lithium hydroxide monohydrate (1.2 g, 28.4 mmol)
was dissolved in H2O (28 mL), and the solution was added to the
reaction mixture over 5 min. The solution was allowed to warm to
room temperature and stirred for 5 h. The mixture was diluted with
H2O (45 mL) and extracted with ethyl ether (3 × 50 mL). The aqueous
layer was acidified with aqueous 3 N HCl to pH 1. The solution was
extracted with ethyl acetate (5 × 70 mL). The combined organic phases
were dried over Na2SO4, concentrated, and dried under vacuum to yield
PNA monomers as colorless solids.
Table 1. Mass Characterization Data for All PNAs
entrya
sequence
calculated
Nielsen Sequence
1
2
3
4
5
6
7
8
9
GTAGATCACT-Lys
2855.2
2895.2
2895.2
2895.2
2935.3
2935.3
2975.3
2975.3
3015.3
2895.2
2855.6
2895.9
2895.9
2895.8
2934.7
2934.5
2976.6
2971.1
3016.5
2895.6
GTAGAT*CACT-Lys
GTAGATCA*CT-Lys
GTAGATC*ACT-Lys
GTAGAT*C*ACT-Lys
GT*AGAT*CACT-Lys
GT*AGAT*CA*CT-Lys
GTAGA*T*C*ACT-Lys
GT*AGA*T*CA*CT-Lys
GTAGAT*RCACT-Lysc
10
p53
GGCAGTGCCT-Lys
GGCAGT*GCCT-Lys
11
12
2872.2
2912.2
2873.5
2913.3
a Cyclopentane stereochemistry is (S,S), unless indicated otherwise. B*
) tcyp residue. b PNAs were characterized using a PerSeptive Biosystems
Voyager DE MALDI-TOF system with 2′,4′,6′-trihydroxyacetophenone
monohydrate matrix. Mass spectra were acquired using a N2 laser (337 nm
wavelength, 5 ns pulse), with at least 100 shots per sample. All PNA
oligomers gave molecular ions consistent with the final product. c B*R
tcyp with (R,R) stereochemistry.
)
procedure B, 7 (0.20 g, 0.34 mmol) was converted to 0.19 g (95%) of
PNA monomer 10: mp ) 135 °C (dec); [R]23 ) -30.5° (c ) 1.0,
MeOH, 100%); IR (KBr) 3250, 3196, 3124, 2977, 1750, 1707, 1669,
1615, 1521, 1456, 1214, 1159, 996, 697, 640 cm-1; H NMR (500
D
1
MHz, DMSO) δ major rotamer 10.69 (s, 1H, Cbz-NH), 8.59 (s, 1H,
adenine-H), 8.23 (s, 1H, adenine-H), 7.46 (d, J ) 7.5 Hz, 2H, o-Ph-
H), 7.40 (t, J ) 7.5 Hz, 2H, m-Ph-H), 7.34 (t, J ) 7.5 Hz, 1H, p-Ph-
H), 7.07 (br d, J ) 8.5 Hz, 1H, Boc-NH), 5.40 (q, J ) 17.0 Hz, 2H,
adenine-CH2), 5.22 (s, 2H, Ph-CH2), 4.40-3.78 (m, 4H, NH-CH2-
CO2Me + CH-NH), 2.00-1.40 (m, 6H, cyclopentane-H), 1.39 (s,
9H, tert-butyl-CH3), minor rotamer 6.76 (br d, J ) 8.5 Hz, 1H, Boc-
NH), 1.36 (s, 9H, tert-butyl-CH3); 13C NMR (125 MHz, DMSO) δ
170.5, 166.5, 155.5, 152.5, 152.3, 151.4, 145.1, 136.4, 128.5, 128.0,
127.9, 123.0, 78.0, 66.3, 61.4, 52.8, 44.1, 43.8, 28.2, 25.9, 18.8; HRMS
(ESI-MS m/z) mass calcd for C27H34N7O7 [M + H]+, 568.2520, found
568.2501.
N-[(2S)-tert-Butoxycarbonylaminocyclopent-(1S)-yl]-N-[thymin-
1-ylacetyl]glycine (8). Following general procedure B, 5 (0.93 g, 2.12
mmol) was converted to 0.87 g (96%) of PNA monomer 8: mp ) 203
°C (dec); [R]23 ) -36.5° (c ) 1.0, MeOH, 100%); IR (KBr) 3351,
D
3178, 2976, 2611, 2531, 1682, 1530, 1455, 1365, 1244, 1167, 1045,
1
853, 782, 467 cm-1; H NMR (500 MHz, DMSO) δ major rotamer
12.41 (br s, 1H, CO2H), 11.28 (s, 1H, imide-NH), 7.15 (s, 1H,
thymine-H), 6.94 (d, J ) 8.0 Hz, 1H, Boc-NH), 4.71 (q, J ) 17.0
Hz, 2H, thymine-CH2), 3.9-3.7 (m, 4H, NH-CH2-CO2Me + CH-
NH), 1.9-1.4 (m, 6H, cyclopentane-H), 1.75 (s, 3H, thymine-CH3),
1.36 (s, 9H, tert-butyl-CH3), minor rotamer 6.75 (d, J ) 8.0 Hz, 1H,
Boc-NH), 4.44 (q, J ) 17.0 Hz, 2H, thymine-CH2); 13C NMR (125
MHz, DMSO) δ 177.5, 167.1, 164.4, 155.4, 151.0, 141.8, 108.2, 77.9,
61.2, 52.7, 47.9, 43.8, 28.2, 26.0, 18.9, 12.0; HRMS (ESI-MS m/z)
mass calcd for C29H29N4O7 [M + H]+, 425.2036, found 425.2043.
N-[(2R)-tert-Butoxycarbonylaminocyclopent-(1R)-yl]-N-[thymin-
1-ylacetyl]glycine. Prepared through identical procedures used to
prepare 8 utilizing (R,R)-diamine. All characterization data matched 8
Synthesis of Peptide Nucleic Acids (PNAs). See Supporting
Information for detailed procedures. All PNAs were purified by
reversed-phase HPLC, and were characterized by MALDI-TOF mass
spectrometry as shown in Table 1.
Results
The synthesis of tcypPNAs requires access to significant
quantities of trans-cyclopentane diamine. Our initial, published
synthesis was employed so that cycloalkane rings ranging from
cyclopropane to cyclopentane could be incorporated into the
PNA backbone.12 Since then, cyclopentane has emerged as the
optimal ring size. Therefore, we have focused our synthetic
efforts to exclusively make cyclopentane PNA monomers. A
central limitation of the previous route was the low-yielding
reactions that were required to differentiate between the two
identical nitrogens of trans-1,2-cyclopentane diamine. A more
efficient synthesis yields the same diamine, but with the two
nitrogens introduced in a stepwise fashion onto the five-
membered ring so that orthogonal protecting groups can be
placed on the nitrogens.
Such a route was developed on the basis of recent progress
in cyclopentane â-amino acid synthesis developed by Gellman
and co-workers (Scheme 1).17 Starting from ethyl 2-oxocyclo-
pentanecarboxylate, conversion to the Boc-protected â-amino
acid 1 was accomplished via reductive amination, recrystalli-
zation to a single diastereomer, and deprotection/reprotection
except [R]23 ) +36.5° (c ) 1.0, MeOH, 100%).
D
N-[(2S)-tert-Butoxycarbonylaminocyclopent-(1S)-yl]-N-[4-N-(ben-
zyloxycarbonyl)cytosin-1-ylacetyl]glycine (9). Following general
procedure B, 6 (0.88 g, 1.54 mmol) was converted to 0.67 g (80%) of
PNA monomer 9: mp ) 155 °C (dec); [R]23 ) -35.0° (c ) 1.0,
D
MeOH, 100%); IR (KBr) 3359, 3326, 2972, 1750, 1668, 1497, 1455,
1
1367, 1215, 1064, 1045, 1002, 783, 745, 695 cm-1; H NMR (500
MHz, DMSO) δ major rotamer 12.35 (br s, 1H, CO2H), 10.78 (br s,
1H, Cbz-NH), 7.76 (d, J ) 7.0 Hz, 1H, cytosine-H), 7.41 (m, 5H,
Ph-H), 7.01 (d, J ) 7.0 Hz, 1H, cytosine-H), 6.94 (d, J ) 7.5 Hz,
1H, Boc-NH), 5.19 (s, 2H, Ph-CH2), 4.89 (q, J ) 16.0 Hz, 2H,
cytosine-CH2), 4.3-3.7 (m, 4H, NH-CH2-CO2H + CH-NH), 1.9-
1.4 (m, 6H, cyclopentane-H), 1.36 (s, 9H, tert-butyl-CH3), minor
rotamer 7.80 (d, J ) 7.0 Hz, 1H, cytosine-H), 6.74 (d, J ) 7.5 Hz,
1H, Boc-NH), 4.60 (q, J ) 16.0 Hz, 2H, cytosine-CH2); 13C NMR
(125 MHz, DMSO) δ 171.2, 170.5, 167.0, 162.9, 155.4, 154.7, 153.2,
150.7, 136.0, 128.5, 128.2, 128.0, 93.9, 77.9, 66.6, 61.1, 52.8, 49.6,
43.8, 28.2, 26.1, 18.9; HRMS (ESI-MS m/z) mass calcd for C26H34N5O8
[M + H]+, 544.2407, found 544.2405.
N-[(2S)-tert-Butoxycarbonylaminocyclopent-(1S)-yl]-N-[6-N-(ben-
zyloxycarbonyl)adenin-9-ylacetyl]glycine (10). Following general
9
15070 J. AM. CHEM. SOC. VOL. 126, NO. 46, 2004

(S,S)-trans-Cyclopentane-Constrained Peptide Nucleic Acids
A R T I C L E S
Scheme 1
Table 2. Melting Temperature Data for PNA-DNA Complexes
following the published procedures. From 1, a Curtius rear-
rangement, followed by trapping the intermediate isocyanate 2
with benzyl alcohol, in the presence of stoichiometric CuCl as
a Lewis acid activator,19 afforded cyclopentane diamine 3, in
which the two nitrogens are conveniently protected with
orthogonal protecting groups. Deprotection of the Cbz-protected
nitrogen, followed by alkylation with a bromoacetate ester,
afforded 4, the essential backbone to make tcypPNA monomers.
From this intermediate, all four DNA bases were attached using
established procedures for the synthesis of PNA monomers. In
this paper, we report the synthesis and complete characterization
of cyclopentane PNA monomers with thymine, adenine, and
cytosine. Therefore, esters 5, 6, and 7 were synthesized, and
ester hydrolysis afforded tcypPNA monomers 8, 9, and 10 that
were used directly in solid-phase peptide synthesis. The
synthesis of the corresponding tcypPNA monomer with guanine
was particularly challenging, and involved new chemistry that
will be the subject of a future publication.
Using modified manual solid-phase peptide synthesis proce-
dures,21 12 different PNAs were synthesized to probe the effects
of cyclopentane incorporation into PNA. In general, cyclopen-
tane PNA monomers could be readily inserted into a PNA
sequence one or multiple times without difficulty (see Support-
ing Information for details). After cleavage from the resin, each
PNA was readily purified by reversed-phase HPLC and
characterized by MALDI-TOF mass spectrometry. Even though
addition of cyclopentanes into a PNA should increase the
hydrophobicity of the PNA and could reduce aqueous solubility,
all the synthesized tcypPNAs were completely soluble under
the aqueous conditions used in this work (up to 300 µM).
The effects of trans-cyclopentane incorporation on PNA-
DNA stability were studied within a 10-residue, mixed-base
PNA sequence that has been extensively studied in the literature
(by Nielsen and co-workers in particular).7,22 Data from variable-
temperature UV absorbance were used to determine the influ-
ence of trans-cyclopentane on binding affinity relative to
entrya
sequence
Tm
(°
C) for DNAb
∆Tm (°
C)c
Nielsen Sequence
1
2
3
4
5
6
7
8
9
GTAGATCACT-Lys
48.9
54.9
54.5
54.2
60.2
59.6
63.2
64.4
70.3
nae
GTAGAT*CACT-Lys
GTAGATCA*CT-Lys
GTAGATC*ACT-Lys
GTAGAT*C*ACT-Lys
GT*AGAT*CACT-Lys
GT*AGAT*CA*CT-Lys
GTAGA*T*C*ACT-Lys
GT*AGA*T*CA*CT-Lys
GTAGAT*RCACT-Lysd
6.0
5.6
5.3
11.3
10.7
14.3
15.5
21.4
na
10
p53
GGCAGTGCCT-Lys
GGCAGT*GCCT-Lys
11
12
57.7
65.7
8.0
a Cyclopentane stereochemistry is (S,S), unless indicated otherwise. B*
) tcyp residue. b Conditions for Tm measurement: 150 mM NaCl, 10 mM
phosphate buffer, pH 7.0, 0.1 mM EDTA, UV measured at 260 nm from
90 to 25 °C, in 1 °C increments. All values are averages from two or more
experiments. Approximate error for Tm’s is (0.6 °C. c ∆Tm is the difference
in melting temperature between unmodified PNA (entry 1) and cyclopentane
modified PNA, unless indicated otherwise. d B*R ) tcyp with (R,R)
stereochemistry e No detectable melting transition observed.
unmodified PNA. Incorporation of one (S,S)-trans-cyclopentane
into several different positions within this PNA sequence results
in an increase in the melting temperature (Tm) when bound to
complementary DNA (Table 2, entries 1-4). Importantly, this
increase is consistent regardless of the sequence position or
nucleobase composition of the cyclopentane-modified residue.
Furthermore, the benefit to binding is additive when multiple
cyclopentanes are incorporated (Table 2, entries 5-9, Figure
2). Based on the increasing Tm’s, each cyclopentane in the PNA
increases the Tm by an average value of +5 °C.
The stereochemistry of the cyclopentane ring is critical to
attain improved stability in the PNA-DNA duplex. Increases
in Tm’s are clearly observed when the stereochemistry of the
cyclopentane ring is (S,S). In contrast, there is no well-defined
melting transition in the UV analysis if the stereochemistry is
changed to (R,R) (entry 10).
(21) Koch, T.; Hansen, H. F.; Andersen, P.; Larsen, T.; Batz, H. G.; Otteson,
K.; Orum, H. J. Pept. Res. 1997, 49, 80.
(22) (a) Tomac, S.; Sarkar, M.; Ratilainen, T.; Wittung, P.; Nielsen, P. E.;
Norden, B.; Graslund, A. J. Am. Chem. Soc. 1996, 118, 5544. (b) Ratilainen,
T.; Holmen, A.; Tuite, E.; Haaima, G.; Christensen, L.; Nielsen, P. E.;
Norden, B. Biochemistry 1998, 37, 12331.
A biologically relevant sequence was also investigated to
determine whether the cyclopentane modification is sequence
independent. A PNA sequence was constructed that is comple-
mentary to a portion of DNA encoding a mutant p53 protein
9
J. AM. CHEM. SOC. VOL. 126, NO. 46, 2004 15071

A R T I C L E S
Pokorski et al.
Table 4. Discrimination of Single Base Mismatches and
Improvements in tcypPNA
b
Tm
(∆Tm)
entrya
sequence
TT mismatch
TC mismatch
TG mismatch
1
2
6
7
8
9
GTAGATCACT-Lys
GTAGAT*CACT-Lys
GT*AGAT*CACT-Lys
GT*AGAT*CA*CT-Lys 36.5 (-26.7) 30.1 (-33.0) 38.6 (-24.5)
GTAGA*T*C*ACT-Lys 37.8 (-26.6) 29.5 (-34.9) 40.7 (-23.7)
GT*AGA*T*CA*CT-Lys 38.8 (-31.5) 32.3 (-38.0) 42.8 (-27.5)
36.0 (-12.9) 32.0 (-16.9) 34.0 (-14.9)
34.7 (-20.5) 29.4 (-25.8) 36.8 (-18.3)
42.5 (-17.1) 29.6 (-29.5) 40.1 (-19.7)
a Entry numbers correlate to the entries in Table 1 with identical PNA
sequences. B* ) tcyp residue with (S,S) stereochemistry. b All values
reported in units of °C. ∆Tm represents the difference in melting temperature
between the complementary DNA and the DNA with the indicated
mismatch. All mismatches were opposite to residue symbolized with B or
B*.
DNA duplex, and that this stability is largely derived from more
favorable entropy of binding.
Figure 2. Calculated fraction of PNA duplexes as a function of temperature,
with zero (A) to four (E) (S,S)-cyclopentanes incorporated into the PNA
sequence. The curves represent the indicated sequences listed in Table 1:
A ) entry 1; B ) entry 2; C ) entry 5; D ) entry 8; E ) entry 9.
To properly identify SNPs, any DNA probe must be very
effective at discriminating between single base mismatches.
Therefore, the sequence specificity of cyclopentane-modified
PNA was examined by determining the change in Tm of a
DNA-PNA duplex when a single base mismatch is present.
Incorporation of (S,S)-trans-cyclopentane residues generally
improves the mismatch discrimination when using the Nielsen
PNA sequence (Table 4). The mismatches were positioned
opposite to a cyclopentane residue at the sixth position in the
sequence, and a measure of the sequence specificity was
determined by taking the difference in the melting temperature
between the fully matched DNA-PNA duplex and the duplex
with a single base mismatch (this is expressed as ∆Tm in Table
4). One trans-cyclopentane residue in the Nielsen sequence was
found to improve mismatch discrimination over unmodified
PNA, and each subsequent addition of cyclopentane residues
further improves discrimination (except in the case of entry 6
when studied in the context of the TT mismatch). The fact that
this trend is observed for a TG mismatch is of particular
importance, as this mismatch is generally considered to be the
most difficult to discriminate against due to the facile formation
of a wobble base pair.24 The large differences in melting
temperatures observed for tcypPNAs bound to complementary
vs noncomplementary DNA allows one to control hybridization
conditions to exclude binding of noncomplementary DNA by
adjusting the temperature.
Table 3. Thermodynamic Parameters for tcypPNA (entry 8, Table
1) Compared to aegPNA
∆
H
∆
(cal/mol
S
∆G
(kcal/mol)b
entrya
sequence
(kcal/mol)b
‚
K)b
1
8
GTAGATCACT-Lys
GTAGA*T*C*ACT-Lys -66 ( 4 -170 ( 9
-74 ( 9 -203 ( 25 -13 ( 9
-15 ( 4
a Entry numbers correlate to the entries in Table 1 with identical PNA
sequences. B* ) tcyp residue with (S,S) stereochemistry. b Values are
calculated from a line fit to a plot of 1/Tm vs ln(Ct). Errors are calculated
on the basis of the line fit. ∆S and ∆G are calculated at T ) 298 K.
that is prevalent in many different cancer cell lines. Specifically,
the PNA sequence is complementary to the G-to-A mutation at
codon 175 of the p53 protein.23 Recognition of such a DNA
sequence could become an important component for diagnosis
of specific types of cancer. As shown for the previous PNA
sequences, a consistent increase in the Tm was observed (entry
12). Taken together, these data show the general benefits of
cyclopentane incorporation to the stability of PNA-DNA
duplexes. Such benefits should offer more sensitive probes that
can be used in diagnostic assays for DNA.
Several additional characteristics of the cyclopentane modi-
fication have also been investigated relative to unmodified PNA.
A Job plot indicates that a tcypPNA with three cyclopentanes
(entry 8, Table 2) has a 1:1 stoichiometry when bound to
complementary DNA. In addition, both tcypPNA and the
unmodified PNA have similar single-stranded melting profiles,
and the degrees of hysteresis for heating and cooling runs in
single strand and in PNA-DNA duplex melting experiments
are similar (see Supporting Information for the data).
To understand how cyclopentane improves binding affinity,
the thermodynamic values of binding to DNA were determined
for PNA vs a tcypPNA. Variable-concentration Tm data were
obtained for each PNA-DNA duplex and subjected to a Van’t
Hoff analysis. The calculated thermodynamic values for binding
are presented in Table 3 (see Supporting Information for the
data). These data indicate that each cyclopentane contributes
approximately 0.7 kcal/mol to the overall stability of the PNA-
One of the benefits that PNA can offer to DNA detection is
the ability to hybridize to DNA under low salt conditions. Under
such conditions, DNA secondary structure can be denatured10
so that all DNA binding sites are available for detection.
Typically, PNAs bind with higher affinity at lower salt
concentrations than at higher concentrations. For the tcypPNAs
in Table 5, this trend is similar to that observed for the
aegPNAs.22a
Discussion
In this paper, we have reported the benefits of incorporating
trans-cyclopentane units into an aegPNA backbone. The tcyp-
PNAs bind with higher affinity and better sequence specificity
compared to unmodified PNA, and the incorporation of multiple
cyclopentanes improves these properties further. From variable-
(23) Hollstein, M.; Rice, K.; Greenblatt, M. S.; Soussi, T.; Fuchs, R.; Sorlie,
T.; Hovig, E.; Smith-Sorensen, B.; Montesano, R.; Harris, C. C. Nucleic
Acids Res. 1994, 22, 3551.
(24) (a) Allawi, H. T.; SantaLucia, J. Biochemistry 1997, 36, 10581. (b) Igloi,
G. L. P. Natl. Acad. Sci. U.S.A. 1998, 95, 8562.
9
15072 J. AM. CHEM. SOC. VOL. 126, NO. 46, 2004

(S,S)-trans-Cyclopentane-Constrained Peptide Nucleic Acids
A R T I C L E S
Table 5. Ionic Effects on the Thermal Stability (Tm) of Modified
PNAs
cyclohexane is most likely due to conformational differences
between the two rings. The recent success with cis-cyclopentane
in stabilizing oligothymine PNA2-DNA triplexes indicates that
the cis stereochemistry about the cyclopentane ring is also
compatible with the requisite conformations to promote PNA
binding to DNA.14e,i However, further work will be required to
determine whether the cis- or trans-cyclopentane stereochemistry
is the most beneficial in terms of stability and sequence
specificity for a large number of different PNA-DNA duplexes.
The properties of tcypPNAs could impact the design of new
probes for genomic analysis. DNA detection with PNA-based
systems could lead to new diagnostics that are more sensitive,
faster, and more durable than the current state-of-the-art systems.
Clearly, the standard aegPNAs have not replaced DNA oligo-
mers as hybridization probes in most diagnostic applications,
most likely due to limitations in the properties and the synthesis
of aegPNAs. Furthermore, there are currently no standard
backbone modifications available that can be used to adjust the
binding properties of aegPNA. In contrast, there are a large
number of modified analogues of the natural nucleosides (such
as the locked nucleic acids) that are routinely used to fine-tune
the binding properties of DNA and RNA oligomers for different
applications.31 The development of new PNA monomers that
improve the binding properties of aegPNAs could spark the
widespread use of such oligomers in a wide variety of
applications. The tcypPNAs described in this paper allow one
to tune the oligomers’ DNA binding properties by calculating
the number of cyclopentanes required. In particular, using these
molecules as DNA binding probes could make current diagnostic
techniques significantly more reliable. We are currently examin-
ing the benefits of using tcypPNAs in PCR-32 and microarray-
based applications.
[NaCl]b
entrya
sequence
0 mM
150 mM
300 mM
2
4
GTAGAT*CACT-Lys
GTAGATC*ACT-Lys
60.8
60.1
55.1
54.2
53.2
52.2
a Entry numbers correlate to the entries in Table 1 with identical PNA
sequences. B* ) tcyp residue. All Tm values reported in units of °C. b [NaCl]
refers to the amount of exogenous salt added to 10 mM sodium phosphate
buffer.
concentration Tm data, the increase in DNA binding affinity of
a cyclopentane-modified PNA is due largely to an entropic
effect, indicating that replacing the ethylenediamine portion of
a PNA with a carbocyclic ring significantly reduces the
conformational flexibility of an unbound PNA, lowering the
entropic cost associated with formation of a PNA-DNA
complex.25 The enthalpy of binding calculated from these data
is consistent with a tcypPNA-DNA duplex that is similar in
structure to an aegPNA-DNA duplex.22a,26
The stereochemistry and size of the ring incorporated at this
position also impact the ability of PNA to bind DNA. For
instance, previously published studies that examined the effects
of replacing the ethylenediamine portions of PNAs with trans-
cyclohexane did not report the same benefits as we report for
trans-cyclopentane.27 Furthermore, both studies have found that
the (S,S) stereochemistry in five- and six-membered rings is
more compatible than (R,R) in the PNA-DNA duplex. We
speculate that, in order for a carbocyclic ring to promote PNA
binding to oligonucleotides, both ring size and stereochemistry
must restrict the PNA to access only the range of dihedral angles
necessary for binding, while excluding conformations that are
irrelevant to duplex formation. In a PNA-DNA duplex, the
dihedral angles about the ethylenediamine units adopt a range
of values depending on the position of each unit in the sequence.
From an NMR-derived PNA-DNA structure, the approximate
range for this dihedral angle is 130-165°.28 In crystal structures,
the same dihedral angle has an average value of 73° in the
Watson-Crick portion of a PNA2-DNA triplex,29 and a range
of 60-83° in a D-Lys-modified PNA-DNA duplex.30 In our
previous publication, we used molecular modeling to demon-
strate that the conformations of a five-membered ring should
allow PNAs to access the requisite conformations for DNA
binding to a greater extent than a six-membered ring.12
Therefore, the success of trans-cyclopentane compared to trans-
Acknowledgment. We gratefully acknowledge financial
support from Northwestern University’s Weinberg College of
Arts and Sciences, Department of Chemistry, VP of Research,
and the Institute of Bioengineering and Nanoscience in Ad-
vanced Medicine (IBNAM). J.K.P. was supported by North-
western University’s NIH Biotechnology Training Grant (T32
GM008449).
Supporting Information Available: Procedures for solid-
phase synthesis of all PNAs, mass spectra to characterize all
PNAs, 1H and 13C spectra for all compounds listed in the
Experimental Section, data for single-strand melting experi-
ments, hysteresis between heating and cooling melting experi-
ments, plot of 1/Tm vs ln Ct from variable-concentration melting
temperature data, and Job plots. This material is available free
of charge via the Internet at http://pubs.acs.org.
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