Cyclopropane PNA: Observable triplex melting in a PNA constrained with a 3-membered ring

Article abstract of DOI:10.1016/j.tetlet.2004.12.061

The first peptide nucleic acid (PNA) with a cyclopropane in the backbone has been synthesized, and the effects of the ring on DNA/RNA binding properties of the PNA have been examined. Well-defined triplex to duplex melting transitions of PNA₂ DNA complexes is clearly observed by variable temperature UV absorbance with the cyclopropane-constrained PNA.

Full text of DOI:10.1016/j.tetlet.2004.12.061

Tetrahedron
Letters
Tetrahedron Letters 46 (2005) 915–917
Cyclopropane PNA: observable triplex melting in a
PNA constrained with a 3-membered ring
Jonathan K. Pokorski, Michael C. Myers and Daniel H. Appella*
Northwestern University, Department of Chemistry, 2145 Sheridan Road, Evanston, IL 60208, USA
Received 17 November 2004; revised 10 December 2004; accepted 14 December 2004
Available online 29 December 2004
Abstract—The first peptide nucleic acid (PNA) with a cyclopropane in the backbone has been synthesized, and the effects of the ring
on DNA/RNA binding properties of the PNA have been examined. Well-defined triplex to duplex melting transitions of PNA2
DNA complexes is clearly observed by variable temperature UV absorbance with the cyclopropane-constrained PNA.
Ó 2004 Elsevier Ltd. All rights reserved.
Peptide nucleic acids (PNAs) are nucleic acid mimics
that possess a polyamide backbone instead of the su-
gar–phosphate backbone of DNA/RNA. The most com-
mon PNA is derived from an (aminoethyl)glycine (aeg)
backbone which was first introduced by Nielsen et al.
(Fig. 1A).1 The aegPNAs bind to complementary
DNA and RNA sequences through Watson–Crick
(and in some cases Hoogsteen) hydrogen bonding with
higher affinity than the corresponding DNA or RNA se-
quences.2 Since the introduction of aegPNA, researchers
have attempted to modify the simple polyamide back-
bone to study the effects of different modifications on
the oligonucleotide binding properties.3 Recent efforts
in this area have focused on the incorporation of cyclic
rings into the PNA backbone in attempts to pre-orga-
nize the PNA for oligonucleotide binding.4 The most
successful modifications using cyclic restraints involve
the introduction of 5- or 6-membered rings into various
positions in the backbone.5,6 Smaller rings have, to the
best of our knowledge, not been explored for their ef-
fects on PNA binding to oligonucleotides. In previous
work, we have shown that the introduction of trans-
cyclopentane rings into a PNA backbone affords a class
of PNAs (which we refer to as tcypPNAs) with both
higher binding affinity and improved sequence specific-
ity to complementary DNA (Fig. 1B).6 To probe the
effects of smaller carbocyclic rings on the oligonucleo-
tide-binding properties of PNA, we have incorporated
a trans-cyclopropane ring into the PNA backbone
(Fig. 1C). Compared to cyclopentane, the cyclopropane
ring is more rigid, and the dihedral angle between trans
substituents on the 3-membered ring (approximately
145°) is larger than the corresponding 5-membered ring
(approximately 70°).6a,7 Herein, we report an asymmet-
ric synthesis of the trans-cyclopropane (tcpr) PNA
monomer and binding studies of a poly-T heptamer with
one cyclopropane monomer to complementary DNA
and RNA.
Base
O
Base
O
O
O
O
O
N
N
N
Base
The tcprPNA monomer was synthesized as shown in
Scheme 1. The synthesis began by making (À)-dimen-
thyl succinate (1) fromsuccinic anhydride and ( À)-men-
thol under Dean–Stark conditions. Then, YamamotoÕs
asymmetric alkylation of (À)-dimenthyl succinate with
bromochloromethane afforded the (S,S)-cyclopropane
dimenthyl ester 2 in 45% yield after recrystallization
(>99% de).8 Hydrolysis of 2 under basic conditions, fol-
lowing YamamotoÕs procedure, gave (1S,2S)-cyclopro-
pane-1,2-dicarboxylate (3) as a single enantiomer. A
N
H
N
HN
H
(A)
(B)
(C)
Figure 1. (A) aegPNA, (B) tcypPNA, (C) tcprPNA.
Keywords: Peptide nucleic acid; PNA; Cyclopropane.
*
Corresponding author. Tel.: +1 847 4675963; fax: +1 847 4914813;
e-mail: dappella@chem.northwestern.edu
0040-4039/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2004.12.061

916
J. K. Pokorski et al. / Tetrahedron Letters 46 (2005) 915–917
O
O
O
O
Me
Me
O
HO
+
OMen
OMen
OH
OH
ii. (a-c)
45%
OMen
OMen
i.
iii.
O
83%
95%, >99%ee
O
O
1
O
Me
3
2
(-)-Menthol
(Men)
NHBoc
NHBoc
NHBoc
NHBoc
NH2
iv. (a-d)
75%
v.
vi.
vii.
OMe
N
77%
90%
OMe
42%
N
H
NHBoc
O
O
O
7
4
5
6
T
NHBoc
O
viii.
Me
OH
NH
96%
N
T =
O
N
O
O
T
8
Scheme 1. Reagents and conditions: (i) cat. p-TsOH, toluene, Dean stark, 18 h; (ii) (a) LiTMP, À78 °C; (b) BrClCH2; (c) terephthalaldehyde; (iii)
10% KOH, 9:1 MeOH/H2O, 60 °C, 4 h; (iv) (a) EtOCOCl, TEA; (b) NaN3, H2O; (c) benzene, reflux; (d) t-butanol, reflux, 15 h; (v) 20 equiv TFA,
Et2O, 12 h; (vi) methylbromoacetate, DIEA, DMF, 15 h; (vii) T-CH2COOH, HATU, DIEA, 24 h; (viii) LiOH, THF, H2O, 4 h.
Curtius rearrangement, followed by trapping the
intermediate isocyanate with tert-butanol, gave the
di-protected tert-butyl carbamate 4 in 75% yield.
Mono-deprotection of 4 with trifluoroacetic acid gave
mono-amine 5 in 42% yield with a 30% recovery of start-
ing material which could be resubmitted to the reaction
conditions. Mono-amine 5 was alkylated with methyl-
bromoacetate to give 6, and then thymine acetic acid
was coupled to 6 using HATU to give 7. Hydrolysis of
7 afforded (S,S)-tcprPNA monomer 8. This route
yielded tcprPNA monomer in eight steps with a 9%
overall yield and allowed access to gramquantities of
material. The cyclopropane monomer was then incorpo-
rated into a PNA oligomer using modified solid phase
peptide synthesis procedures.9 All oligomers were
purified by reverse phase HPLC and characterized by
MALDI-TOF mass spectrometry (See supporting infor-
mation for procedures and characterization data).
Table 1. Melting data for PNA: Oligonucleotide complexes. All
samples were prepared in 10 mM phosphate buffer (pH 7) containing
150 mM NaCl and 0.1 mM EDTA
Entry
Sequence
PNA2–DNAa
PNA–RNAb
Tump
Tdmn
Tump
Tdmn
(°C)c
(°C)d
(°C)c
(°C)d
9
10
TTTTTTT-Lys
TTTTcprTTT-Lys
44.8
21.6,
46.5
44.8
22.4,
39.0
54.2
49.2
53.5
33.0
a Each strand concentration is 7 lM.
b Each strand concentration is 15 lM.
c Tump refers to a melting run going from 20–80 °C in which readings
were taken in 1 °C increments and held at the temperature for 5 min
before readings were taken.
d Tdmn refers to the reverse run with the same conditions as (c).
The melting curves of the tcprPNA–DNA complex were
more interesting. First, a Job plot confirmed that
tcprPNA 10 formed a 2:1 PNA–DNA triplex.10 Surpris-
ingly, the melting curves of this complex clearly show
two thermal melting transitions (Fig. 2B). We speculate
that the lower transition corresponds to the dissociation
of the Hoogsteen strand of the triplex, while the higher
transition is the melting of the duplex. Analogous bipha-
sic transitions have been seen in triplex melting curves,
but they are rarely seen in the melting of PNA tri-
plexes.11 For instance, the melting of aegPNA 9 shows
only a single broad transition, even though a similar tri-
plex is formed. The TmÕs corresponding to the melting of
the tcprPNA–DNA duplex (46.5 °C for heating, 39.0 °C
for cooling) show significant hysteresis and are similar
to the TmÕs of the corresponding complex with aegPNA,
indicating that the cyclopropane constraint has little ef-
fect on duplex stability. In contrast, the triplex TmÕs
(21.6 °C for heating, 22.4 °C for cooling) show signifi-
cantly less hysteresis, which could signify that the cyclo-
The oligonucleotide binding properties of tcprPNA
heptamer 10 were compared to the corresponding aegP-
NA 9 using variable temperature UV experiments to
examine the melting temperatures (TmÕs) of the com-
plexes. The results indicate that introduction of an
(S,S)-cyclopropane into the PNA backbone lowers the
TmÕs of the DNA and RNA complexes (Table 1). In
the case of the tcprPNA–RNA complex, a well-defined
melting transition was clearly visible in both the heating
and cooling experiments. However, a comparison of the
two experiments shows very significant hysteresis
(Fig. 2A). The calculated Tm fromthe heating experi-
ment was nearly identical to that of aegPNA; however,
the calculated Tm for the cooling experiment was
16 °C lower. Since this hysteresis was not observed in
the heating and cooling runs of the aegPNA–RNA com-
plex, it is possible that tcprPNA binds to RNA more
slowly than aegPNA.

J. K. Pokorski et al. / Tetrahedron Letters 46 (2005) 915–917
917
1.2
1.2
1
(B)
(A)
1
Cooling
Heating
0.8
0.8
0.6
0.4
0.2
0
Cooling
Heating
0.6
0.4
0.2
0
20
30
40
Temperature
70
0
10
50
60
80
0
10
20
30
40
50
60
70
Temperature (°C)
Figure 2. Heating and cooling curves of tcprPNA–oligonucleotide complexes. (A) tcprPNA–RNA, (B) tcprPNA2–DNA.
propane constraint is more compatible in the Hoogsteen
strand of a PNA2DNA triplex than in the Watson–
Crick strand. Unfortunately, no triplex melting was ob-
served in the aegPNA control, so no direct comparison
is currently possible.
ysis. Mass spectra to characterize PNAs, 1H spectra
for all new compounds, and Job plot are also included.
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/
j.tetlet.2004.12.061.
In conclusion, we have made the first PNA with an
(S,S)-trans-cyclopropane in the backbone, and we pres-
ent initial data indicating how this constraint may be
useful to the preorganization of a PNA for oligonucleo-
tide binding. A central premise in our research is 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 dihe-
dral angles necessary for binding, while excluding
conformations that are irrelevant to duplex formation.
Fromthis work, there are indications that the range of
dihedral angles accessible to a cyclopropane ring are
more compatible in the Hoogsteen strand of a PNA2D-
NA triplex than in the Watson–Crick strand. Future
work will focus on examination of tcprPNA in
Hoogsteen interactions within a PNA–tcprPNA–DNA
triplex.
References and notes
1. Nielsen, P. E.; Egholm, M.; Berg, R. H.; Buchardt, O. B.
Science 1991, 254, 1497.
2. (a) Nielsen, P. E. Acc. Chem. Res. 1999, 32, 624; (b)
Nielsen, P. E. Mol. Biotechnol. 2004, 26, 233.
3. Ganesh, K. N.; Nielsen, P. E. Curr. Org. Chem. 2000, 4,
931.
4. Kumar, V. A. Eur. J. Org. Chem. 2002, 2021, and
references therein.
5. (a) Lagriffoule, P.; Wittung, P.; Eriksson, M.; Jensen, K.
K.; Norden, B.; Buchardt, O.; Nielsen, P. E. Chem. Eur. J.
1997, 3, 912; (b) Govindaraju, T.; Gonnade, R. G.;
Bhadbhade, M. M.; Kumar, V. A.; Ganesh, K. N. Org.
Lett. 2003, 5, 3013; (c) Govindaraju, T.; Kumar, V. A.;
Ganesh, K. N. J. Org. Chem. 2004, 69, 1858; (d)
Govindaraju, T.; Kumar, V. A.; Ganesh, K. N. Chem.
Commun. 2004, 860; (e) Govindaraju, T.; Kumar, V. A.;
Ganesh, K. N. J. Org. Chem. 2004, 69, 5725.
6. (a) Myers, M. C.; Witschi, M. W.; Larionova, N. V.;
Franck, J. M.; Haynes, R. D.; Hara, T.; Grajkowski, A.;
Appella, D. H. Org. Lett. 2003, 5, 2695; (b) Pokorski, J.
K.; Witschi, M. A.; Purnell, B. L.; Appella, D. H. J. Am.
Chem. Soc. 2004, 126, 15067.
7. Franck, J. M.; Appella, D. H. Unpublished results.
8. (a) Misumi, A.; Iwanaga, K.; Furuta, K.; Tamamoto, H.
J. Am. Chem. Soc. 1985, 107, 3343; (b) Furuta, K.;
Iwanaga, K.; Yamamoto, H. Org. Synth. 1989, 67, 76.
9. Koch, T.; Hansen, H. F.; Andersen, P.; Larsen, T.; Batz,
H. G.; Otteson, K.; Orum, H. J. Peptide Res. 1997, 49, 80.
10. Job, P. Ann. Chim. 1928, 9, 113.
Acknowledgements
We gratefully acknowledge financial support from
Northwestern UniversityÕs Weinberg College of Arts
and Sciences, Department of Chemistry, and the VP of
Research; and the American Chemical Society Petro-
leumResearch Fund. Jon Pokorski was supported by
Northwestern UniversityÕs NIH Biotechnology Training
Grant (T32 GM008449).
11. (a) Pilch, D. S.; Levenson, C.; Shafer, R. H. Proc. Natl.
Acad. Sci. 1990, 87, 1942; (b) Griffith, M. C.; Risen, L. M.;
Greig, M. J.; Lesnik, E. L.; Sprankle, K. G.; Griffey, R.
H.; Kiely, J. S.; Freier, S. M. J. Am. Chem. Soc. 1995, 117,
831.
Supplementary data
Procedures for synthesis of tcprPNA monomers, solid
phase synthesis of all PNAs, and thermal melting anal-