Hybridization properties of aromatic peptide nucleic acids: A novel class of oligonucleotide analogues

Article abstract of DOI:10.1021/ol010231q

(matrix presented) The synthesis of APNA-PNA chimeras containing all four DNA bases will be described. The hybridization properties of APNA-PNA chimeras with DNA and RNA are consistent with plausible intramolecular interactions that could preorganize the

Full text of DOI:10.1021/ol010231q

ORGANIC
LETTERS
2002
Vol. 4, No. 1
63-66
Hybridization Properties of Aromatic
Peptide Nucleic Acids: A Novel Class
of Oligonucleotide Analogues
,†,‡
Lee D. Fader† and Youla S. Tsantrizos*
Departments of Chemistry, McGill UniVersity, Montreal, Quebec, Canada H3A 2K6,
and Boehringer Ingelheim (Canada) Ltd., LaVal, Quebec, Canada H7S 2G5
ytsantrizos@laV.boehringer-ingelheim.com
Received October 12, 2001
ABSTRACT
The synthesis of APNA-PNA chimeras containing all four DNA bases will be described. The hybridization properties of APNA-PNA chimeras
with DNA and RNA are consistent with plausible intramolecular interactions that could preorganize the oligomers in a duplex or triplex structure
with complementary DNA or RNA.
Peptide nucleic acids (PNAs, I) have great potential as
antisense therapeutics and are known to hybridize with
complementary DNA and RNA with remarkably high affinity
and base-pairing selectivity.1 Early results involving in vitro
a therapeutically practical delivery system. In an attempt to
improve the physicochemical properties of PNAs, a number
of structural analogues have been synthesized.3 Unfortu-
nately, improved cell membrane permeability has not yet
been reported.
From our own studies in this area, we recently reported
the synthesis of the first analogues having an aromatic moiety
as an integral part of the peptidic backbone and we termed
these aromatic peptide nucleic acids (APNA, II4 and III5).
(2) Hanvey, J. C.; Peffer, N. J.; Bisi, J. E.; Thomson, S. A.; Cadila, R.;
Josey, J. A.; Ricca, D. J.; Hassman, C. F.; Bonham, M. A.; Au, K. G.;
Carter, S. G.; Bruckenstein, D. A.; Boyd, A. L.; Noble, S. A.; Babiss, L. E.
Science 1992, 258, 1481.
(3) (a) Bergmeier, S. C.; Fundy, S. L. Bioorg. Med. Chem. Lett. 1997,
7, 3135. (b) Zhang, L.; Min, J.; Zhang, L. Bioorg. Med. Chem. Lett. 1999,
9, 2903. (c) Kuwahara, M.; Arimitsu, M.; Sisido, M. J. Am. Chem. Soc.
1999, 121, 256. (d) Nielsen, P. E. Acc. Chem. Res. 1999, 32, 624. (e) Schu¨tz,
R.; Cantin, M.; Roberts, C.; Greiner, B.; Uhlmann, E.; Leumann, C. Angew.
Chem., Int. Ed. 2000, 39, 1250. (f) Efimov, V. A. Buryakova, A. A.; Choob,
M. V.; Chakhmakcheva, O. G. Nucleosides Nucleotides 1999, 18, 1393.
(g) Puschl, A.; Boesen, T.; Zuccarello, G.; Dahl, O.; Pitsch, S.; Nielsen, P.
E. J. Org. Chem. 2001, 66, 707. (h) Pueschl, A.; Tedeschi, T.; Nielsen, P.
E. Org. Lett. 2000, 2, 4161. (i) D’Costa, M.; Kumar, V.; Ganesh, K. N.
Org. Lett. 2001, 3, 1269. (j) Vilaivan, T.; Suparpprom, C.; Harnyuttanakorn,
P.; Lowe, G. Tetrahedron Lett. 2001, 42, 5533-5536.
cell-free systems or cellular microinjection of PNA oligomers
were encouraging;2 however, their therapeutic utility is
compromised by their inefficient cellular uptake and lack of
† McGill University.
‡ Boehringer Ingelheim (Canada) Ltd.
(1) (a) Nielsen, P. E. Acc. Chem. Res. 1999, 32, 624-630. (b) Nielsen,
P. E. Curr. Opin. Struct. Biol. 1999, 9, 353-357.
10.1021/ol010231q CCC: $22.00 © 2002 American Chemical Society
Published on Web 12/14/2001

A key interest in the design of APNAs was to investigate
the likelihood of forming intramolecular π-staking, or
dipole-quadrapole interactions, along the backbone that
could help in the preorganization of these oligomers and,
consequently, favor duplex or triplex formation with comple-
mentary DNA or RNA. Moreover, we anticipate that the
increased lipophilicity of these analogues would aid their
cell membrane permeability.
oligomerization on solid support. Solution phase couplings
and deprotections of the aniline monomers 5 with the free
acids 1 were followed by deprotection to give the dimers
8a-e (Scheme 2). The subsequent incorporation of dimers
Scheme 2
More recently, we demonstrated that PNA hexamers which
incorporated monomers of general structure III formed stable
triplex structures (albeit less stable than the corresponding
PNA homopolymers) most probably through Watson-Crick
and Ho¨ogsteen base pairing.5 In this Letter, we describe the
synthesis of APNA monomers 1a-d containing all four
natural DNA bases, an optimized procedure for their oligo-
merization into decamers, and preliminary hybridization
properties of their APNA-PNA chimeras with DNA and
RNA.
Synthesis of APNA Monomers and Their Incorporation
into PNA-APNA Chimeras. Methodology that allows for
the efficient preparation of Fmoc-protected APNA monomers
was adapted from our original synthesis of this class of
molecules (Scheme 1, see Supporting Information for syn-
8a-e into the APNA-PNA chimeras 10-12 and 14-16 was
achieved by automated solid-phase synthesis on MBHA resin
(Table 1). The efficiency of coupling at each condensation
Scheme 1a
Table 1. List of Sequences Used in Hybridization Studiesa
PNA
sequence
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
Ac-ATCATTCTCT-Lys-NH2
Ac-ATCAT-TR-CTCT-Lys-NH2
Ac-ATCA-TRTR-CTCT-Lys-NH2
H-ARTRCRAR-TTCTCT-Lys-NH2
H-GTAGATCACT-Lys-NH2
H-GTAGA-TR-CACT-Lys-NH2
H-GTAG-ARTR-CACT-Lys-NH2
H-GRTRARGR-ATCACT-Lys-NH2
H-ATCACT-Lys-NH2
5′-dAGAGAATGAT-3′ (antiparallel)b
5′-rAGAGAAUGAU-3′ (antiparallel)
5′-dAGTGATCTAC-3′ (aniparallel)
5′-dCATCTAGTGA-3′ (parallel)
5′-rAGUGAUCUAC-3′(antiparallel)
5′-rCAUCUAGUGA-3′ (parallel)
5′-dAGTGA-A-CTAC-3′(antiparallel)
5′-dAGTG-T-TCTAC-3′ (antiparallel)
5′-dAGTGATC-A-AC-3′ (antiparallel)
a Conditions: (a) (i) Boc2O/THF, (ii) AllocCl, pyr/DCM, (iii)
HCl/dioxane, 88%, three steps; (b) BrCH2CO2tBu, DIPEA/DMF,
64-71%; (c) BCH2CO2H, HATU, DIPEA/DMF, 76-97%; (d)
Bu3SnH, Pd(PPh3)4, H2O/DCM, 84-86%; (e) FmocCl, Na2CO3,
aq dioxane, 81-89%; (f) TFA/DCM, >97%.
thetic details).5,6 To ensure high overall yields and purity of
the desired APNA homopolymers or APNA-PNA chimeras,
APNA dimers were first synthesized in solution before their
a APNA residues with subscript R and bold text, PNA residues in normal
text. b Orientation defined by the convention introduced by Nielsen et al.
See ref 10.
(4) Tsantrizos, Y. S.; Lunetta, J. F.; Boyd, M.; Fader, L. D.; Wilson,
M.-C. J. Org. Chem. 1997, 62, 5451.
(5) Fader, L. D.; Boyd, M.; Tsantrizos, Y. S. J. Org. Chem. 2001, 66,
3372.
step was monitored by spectrophotometric analysis of the
liberated piperidine-dibenzofulvene adduct.7 A protocol
involving preactivation of each APNA dimer with HATU/
HOAt/2,4,6-collidine, followed by reaction with the aniline
moiety of the resin-bound oligomer (6 h), led to a coupling
(6) (a) For the synthesis of (Cbz)GuCH2CO2H, see: Coull, J. M.; Hodge,
R. P. Patent WO, 95/17403, 1995. (b) For synthesis of (Cbz)AdCH2CO2H
and ThCH2CO2H, see: Thomson, S. A.; Josey, J. A.; Cadilla, R.; Gaul, M.
D.; Hassman, C. F.; Luzzio, M. J.; Pipe, A. J.; Reed, K. L.; Ricca, D. J.;
Wiethe, R. W.; Noble, S. A. Tetrahedron 1995, 51(22), 6179-6194. (c)
(Cbz)CyCH2CO2H was synthesized using an independent method.
64
Org. Lett., Vol. 4, No. 1, 2002

efficiency of >95% for each cycle. The PNA portions of
PNA-APNA chimeras were synthesized from Fmoc/Cbz-
protected monomers, prepared as previously described,6b
followed by oligomerization on solid support using stand-
ard chain elongation reactions.8 The oligomers were
then cleaved from the resin and purified as previously
described.5
Hybridization of APNA-PNA/DNA or APNA-PNA/
RNA Duplexes. Similar experiments were conducted with
APNA-PNA chimeras designed to form duplex structures
with DNA and RNA. A base sequence previously described
by Nielsen and co-workers was chosen so that we could make
direct comparisons to published data (i.e., oligomer 13, Table
3).10,11 In our hands, the PNA decamer 13 gave Tm values of
Hybridization of (APNA-PNA)2:DNA or (APNA-PNA)2:
RNA Triplexes. The hybridization properties of triplexes
between the PNA decamer 9 and complementary DNA or
RNA were examined by Tm measurements and compared to
those of the APNA-PNA chimeras 10-12 (Table 2). The
Table 3. Summary of Tm Values for Complexes Formed
between PNA 13-17 and Oligonucleotides 20-26
a
Tm of PNA strand
target strand
13
14
15
16
17
24
<10
26
Table 2. Summary of Tm Values for Complexes Formed
between PNA 9-12 and DNA 18 or RNA 19
20
21
22
23
24
25
26
54
39
58
42
34b
36
44
40
34
48
31
27
nd
nd
30
26
38
27
nd
17
nd
43
28
45
28
nd
nd
38
a
Tm of PNA strand
20
nd
nd
nd
target strand
9
10
11
12
18
19
34
51
27
40
26
35
29
38
a Duplex concentration ) 4-5 µM. Tm experiments were performed as
described for data presented in Table 2. Buffer conditions: 100 mM NaCl,
10 mM NaH2PO4, 0.1 mM EDTA, pH ) 7.0. b Mismatched base in
compounds 24-26 is indicated in bold text (see Table 1).
a PNA:DNA or PNA:RNA molar ratio was 1:1. Solutions were 4-5 µM
in both PNA and DNA or RNA. Samples were heated from 5 to 95 °C
and/or cooled from 90 to 5 °C at a rate of 0.5 °/min, and the absorbance at
260 nm was monitored as a function of temperature. Tm values are the
maxima of the first derivative plots of the absorbance versus temperature
data. Buffer conditions: 150 mM NaCl, 10 mM NaH2PO4, 0.1 mM EDTA,
pH ) 7.0.
54 °C (antiparallel, 13:21) and 39 °C (parallel, 13:22) with
DNA (Table 3)12 and slightly higher values with RNA (Tm
) 58 °C for antiparallel, 13:23) and (Tm ) 42 °C for parallel,
13:24). Surprisingly, the degree of destabilization observed
upon introduction of a single APNA unit in the middle of
an antiparallel PNA:DNA duplex (Table 3, 14:20) was far
greater than that observed for the PNA2:DNA triplex (Table
2, 10:18). However, the degree of destabilization observed
with the parallel PNA:DNA, antiparallel PNA:RNA and
parallel PNA:RNA duplexes was similar in magnitude to that
observed with the corresponding triplexes reported in Table
2.
Subsequently, the effects of longer APNA inserts into the
APNA-PNA chimeras were examined. Incorporation of two
APNA units (decamer 15) led to further destabilization of
the complexes formed with both DNA (15:20 and 15:21)
and RNA (15:22 and 15:23). However, chimeras 16 which
is composed of four APNA units at the N-terminal of the
decamer did not exhibit the same degree of destabilization
per APNA insert as chimera 15 relative to the control
oligomer 13. These results further support the existence of
some cooperative intraresidue interaction between adjacent
APNA momoners that enhances the stability of the duplexes
hybridization affinities of all the oligomers with comple-
mentary antiparallel RNA strands were always greater than
those observed with the complementary antiparallel DNA
strands, consistent with the known properties of PNAs (Table
1).1 Although the APNA-PNA chimeras 10-12 gave com-
plexes with both DNA and RNA that were thermally less
stable than those of the PNA decamer 9, they were
significantly more stable than the corresponding DNA:DNA
and DNA:RNA complexes under the same conditions (data
not shown). The destabilization observed with chimera 10
was consistent with our previous hybridization results for
this class of monomer.5 Moreover, since APNA-PNA
decamer 12 was found to hybridize equally or more favorably
than the APNA-PNA decamers 10 and 11, it seemed that
multiple insertions of APNA monomers into PNA oligomers
was well tolerated. Job plots9 confirmed a 2:1 stoichiometry
of binding between the PNA strands, or the APNA-PNA
chimeras, and the DNA or RNA oligomers. In addition, the
Tm values showed a dependence on the pH of the solution,
indicating that the complexes formed were most likely tri-
plexes, presumably involving Watson-Crick and Ho¨ogsteen
base pairing.
(10) (a) Hyrup, B.; Egholm, M.; Nielson, P. E.; Wittung, P.; Norde´n,
B.; Buchardt, O. J. Am. Chem. Soc. 1994, 116, 7964. (b) Sforza, S.; Haaima,
G.; Marchelli, R.; Nielsen, P. E. Eur. J. Org. Chem. 1999, 197, 7-204.
(11) In some cases the N-terminal of the oligomers are capped with an
acetate unit in order to prevent acyl transfer of the last nucleobase-
carboxymethyl moiety. However, this measure was not taken with chimeras
14-17 since the sequences studied in ref 13 were not capped in this way.
For comments on the influence a positively charged N-terminal has on the
stability of PNA:DNA complexes, see: Egholm, M.; Buchardt, O.; Nielsen,
P. E.; Berg, R. H. J. Am. Chem. Soc. 1992, 114, 1895.
(7) (a) Chang, C.-D.; Felix, A. M.; Jimenez, M. H.; Meienhofer, J. Int.
J. Pept. Protein Res. 1980, 15, 485. (b) Peptide and Peptidomimetic
Synthesis. Reagents for Drug DiscoVery; Fluka Chemie GmbH: Buchs,
2000; p 123.
(8) (a) Christensen, L.; Fitzpatrick, R.; Gildea, B.; Petersen, K. H.;
Hansen, H. F.; Koch, T.; Egholm, M.; Buchardt, O.; Nielsen, P. E.; Coull,
J.; Berg, R. H. J. Pept. Sci. 1995, 3, 175. (b) Christensen, L.; Fitzpatrick,
R.; Gildea, B.; Petersen, K. H.; Hansen, H. F.; Koch, T.; Egholm, M.;
Buchardt, O.; Nielson, P. E. J. Pept. Sci. 1995, 1(3), 185.
(9) Job, P. Ann. Chim. (Paris) 1928, 9, 113-203.
(12) The Tm value given for this PNA sequence in refs 10a,b was 50 °C
in the case of the antiparallel complex.
Org. Lett., Vol. 4, No. 1, 2002
65

formed. These results are also consistent with the hybridiza-
tion data of the triplexes (Table 2). It should be noted that
well-defined melting curves were obtained for all duplexes
reported in Table 3. The melting curves observed in these
experiments were distinctly different from those observed
by measuring the UV of each corresponding single strand
as a function of temperature. In cases where the Tm of the
transition assigned to the melting of the PNA:DNA or PNA:
RNA duplex was similar to an apparent transition of the
single strand alone, genuine transitions corresponding to
melting of the duplexes could be distinguished by recording
the Tm experiments as a function of both increasing and
decreasing temperature. The single strands showed pro-
nounced hysteresis, whereas the duplexes did not, suggesting
that the sigmoidal curves obtained with the PNA:DNA or
PNA:RNA mixtures are due to the melting of the expected
duplexes. Furthermore, the Tm values of the duplexes formed
between the C-terminal PNA6 segment of the APNA-PNA
chimera 16 (i.e., hexamer 17) with the same complementary
DNA or RNA decamers were found to be significantly lower
than those of decamer 16. The latter results clearly prove
the participation of the N-terminal APNA4 segment of
decamer 16 in the duplexes formed with its complementary
natural oligonucleotides (Table 3).
The introduction of single-point mismatches in the comple-
mentary DNA strands (compounds 24-26) opposite to
APNA residues (chimeras 14-16) led to the anticipated
decrease in the Tm values of the corresponding duplexes as
compared to the fully complementary duplexes (Table 3).
These results strongly support that the APNA monomers
participate in Watson-Crick interactions and contribute to
the overall sequence-specific recognition and binding of
decamers 14, 15, and 16 to complementary DNA and RNA.
It is conceivable that the destabilization observed upon
insertion of an APNA monomer into a PNA oligomer may
be due to a conformational incompatibility and/or distance
between units at the PNA1-APNA-PNA2 junctions; studies
that address this questions are currently in progress.
thiazol-3-ium iodide (DiSC2(5)) binds to the minor groove
of duplexes and triplexes (composed in whole or in part of
PNA oligomers) in a spontaneous and cooperative fashion
as a highly ordered aggregate. The binding is accompanied
by a hypsochromic shift of the main visible absorption band
of the dye, allowing for convenient detection of PNA
containing duplexes or triplexes (see Supporting Information
for the absorption spectra of antiparallel14 duplexes 13:20
and 13:22, Figures S2a and S2b, respectively). Similar results
were obtained with all the complexes formed between
compounds 13-16 and oligonucleotides 20 and 22 (for
example, see figures S2c and S2d for antiparallel duplexes
15:20 and 15:22).15
Although the observed trend in the hybridization properties
of the APNA-PNA chimeras 10-12 and 14-16 cannot be
fully explained by our present data, it appears that continuous
stretches of APNA monomers are well tolerated in both
duplexes and triplexes. In an attempt to evaluate the binding
properties of APNA homopolymers, and any plausible
cooperative stabilization via intraresidue interactions, a
decamer composed of only APNA units was synthesized.
Unfortunately, this molecule was insoluble in aqueous
solvents, prohibiting hybridization studies. The synthesis of
more soluble APNA analogues, composed of heteroaromatic
and substituted phenyl moieties along the backbone, is
currently underway.
Acknowledgment. This work was supported by grants
to Y.S.T. from the Natural Sciences and Engineering
Research Council of Canada. L.D.F. thanks the NSERC
(2000-) for a post graduate fellowship.
Supporting Information Available: 1H, 13C, COSY,
HMQC, and HMBC NMR spectra for compounds 3 and 1a-
1
d, H NMR spectra for dimers 8a-e, MS and HPLC data
for oligomers 9-16, and Tm (first derivative) curves for
thermal denaturation of the complexes indicated in Tables 2
and 3. This material is available free of charge via the Internet
at http//pubs.acs.org.
Duplex formation between compounds 13-17 and oligo-
nucleotides 20 and 22 was also confirmed using the molec-
ular recognition technology developed by Armitage and co-
workers.13 It has been shown that cyanine dye 3-ethyl-2-[5-
(3-ethyl-3H-benzothiazol-2-ylidene)penta-1,3-dienyl]benzo-
OL010231Q
(14) Only the antiparallel complexes were examined for binding to
DiSC2(5). The authors of ref 13 have demonstrated that the dye binds with
very low affinity to parallel PNA complexes.
(15) In a series of control experiments, the UV-vis spectra of compounds
13-17 in the presence of DiSC2(5) were recorded at 40 and then 7 °C, in
the absence of complementary oligonucleotide 20 or 22. No significant
change in the spectra was observed, indicating that a (DiSC2(5))n:ssAPNA-
PNA aggregate was not responsible for the observed spectral changes.
(13) Smith, J. O.; Olson, D. A.; Armitage, B. A. J. Am. Chem. Soc. 1999,
121, 2686-2695.
66
Org. Lett., Vol. 4, No. 1, 2002