Modulating the hybridization property of PNA with a peptoid-like side chain

Article abstract of DOI:10.1021/ol900587b

Modification on the ?-N of the PNA backbone yielded a PNA analogue with a peptoid-like side chain. We found that the length of the side chain was important in influencing the hybridization affinity of the modified PNA.

Full text of DOI:10.1021/ol900587b

ORGANIC
LETTERS
2009
Vol. 11, No. 11
2329-2332
Modulating the Hybridization Property of
PNA with a Peptoid-Like Side Chain
Xiao-Wei Lu, Yun Zeng, and Chuan-Fa Liu*
DiVision of Chemical Biology and Biotechnology, School of Biological Sciences,
Nanyang Technological UniVersity, 60 Nanyang DriVe, Singapore 637551
cfliu@ntu.edu.sg
Received April 6, 2009
ABSTRACT
Modification on the γ-N of the PNA backbone yielded a PNA analogue with a peptoid-like side chain. We found that the length of the side
chain was important in influencing the hybridization affinity of the modified PNA.
PNA is a DNA/RNA mimic with a poly-N-(2-aminoethyl)-
glycine (aeg) backbone (Figure 1a) and binds to comple-
mentary oligonucleotides through Watson-Crick base pair-
ing with high sequence specificity.1,2 The structural simplic-
ity, high hybridization affinity, and in vivo stability of PNA
have made it an attractive agent for antisense and antigene
applications in basic biology and medicine.3-5 Since its
advent in 1991, extensive modification work has been
conducted on PNA in an effort to further improve its
(1) Nielsen, P. E.; Egholm, M.; Berg, R. H.; Buchardt, O. Science 1991,
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(2) Egholm, M.; Buchardt, O.; Christensen, L.; Behrens, C.; Freier, S. M.;
Driver, D. A.; Berg, R. H.; Kim, S. K.; Norden, B.; Nielsen, P. E. Nature
1993, 365, 566
Figure 1. Structures of (a) an aegPNA residue, (b) a peptoid PNA
residue (an aminopeptoid PNA residue, when X ) NH2), and (c)
a protected aminopeptoid PNA monomer for oligomer synthesis.
The red B denotes a nucleobase.
.
(3) (a) Demidov, V. V.; Potaman, V. N.; Frank-Kamenetskii, M. D.;
Egholm, M.; Buchard, O.; Sonnichsen, S. H.; Nielsen, P. E. Biochem.
Pharmacol. 1994, 48, 1310. (b) Kaihatsu, K.; Janowski, B. A.; Corey, D. R.
Chem. Biol. 2004, 11, 749. (c) Nielsen, P. E. Mol. Biotechnol. 2004, 26,
233
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(4) (a) Ray, A.; Norde´n, B. FASEB J. 2000, 14, 1041. (b) Knudsen, H.;
Nielsen, P. E. Anticancer Drugs 1997, 8, 113. (b) Zhilina, Z. V.; Ziemba,
A. J.; Ebbinghaus, S. W. Curr. Top. Med. Chem. 2005, 5, 1119. (c) Seitz,
properties.6-12 These modifications have been mainly fo-
cused on the carbons of PNA’s aeg backbone,6-11 and
analogues with interesting pharmacological properties have
been reported.6-11 Modifications on these backbone carbons
O. Highlights Bioorg. Chem. 2004, 327
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(5) (a) Larsen, H. J.; Bentin, T.; Nielsen, P. E. Biochim. Biophys. Acta
1999, 1489, 159. (b) Janowski, B. A.; Kaihatsu, K.; Huffman, K. E.;
Schwartz, J. C.; Ram, R.; Hardy, D.; Mendelson, C. R.; Corey, D. R. Nat.
Chem. Biol. 2005, 1, 210. (c) Kaihatsu, K.; Janowski, B. A.; Corey, D. R.
Chem. Biol. 2004, 11, 749. (d) Pokorski, J. K.; Nam, J. M.; Vega, R. A.;
Mirkin, C. A.; Appella, D. H. Chem. Commun. 2005, 2101. (e) Ortiz, E.;
Estrada, G.; Lizardi, P. M. Mol. Cell. Probe 1998, 12, 219. (f) Socher, E.;
Jarikote, D. V.; Knoll, A.; Roeglin, A.; Burmeister, J.; Seitz, O. Anal.
Biochem. 2008, 375, 318. (g) Thurley, S.; Roeglin, L.; Seitz, O. J. Am.
Chem. Soc. 2007, 129, 12693. (h) Socher, E.; Bethge, L.; Knoll, A.;
Jungnick, N.; Herrmann, A.; Seitz, O. Angew. Chem., Int. Ed. 2008, 47,
(6) (a) Hyrup, B.; Egholm, M.; Nielsen, P. E.; Wittung, P.; Norden, B.;
Buchardt, O. J. Am. Chem. Soc. 1994, 116, 7964. (b) Nielsen, P. E.; Haaima,
G.; Lohse, A.; Buchardt, O. Angew. Chem., Int. Ed. 1996, 35, 1939
(7) (a) Sforza, S.; Haaima, G.; Marchelli, R.; Nielsen, P. E. Eur. J. Org.
Chem. 1999, 197, 204. (b) Sforza, S.; Corradini, R.; Ghirardi, S.; Dossena,
.
A.; Marchelli, R. Eur. J. Org. Chem. 2000, 2905, 2913
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(8) (a) Zhou, P.; Wang, M.; Du, L.; Fisher, G. W.; Waggoner, A.; Ly,
D. H. J. Am. Chem. Soc. 2003, 125, 6878. (b) Dragulescu-Andrasi, A.;
Zhou, P.; He, G.; Ly, D. H. Chem.Commun. 2005, 244.
9555
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10.1021/ol900587b CCC: $40.75
Published on Web 05/13/2009
 2009 American Chemical Society

Table 1. Thermal Stability Data (Tm, °C) of AP-PNA/DNA and AP-PNA/RNA Duplexesa i
,
a The concentrations of DNA/RNA, aegPNA, and AP-PNA oligomers were 2 µM each for duplex formation in 10 mM phosphate buffer (pH 7)
containing 0.1 M NaCl and 0.1 mM EDTA. Thermal denaturation studies were conducted using UV monitoring at 260 nm scanning from 80 to 20
°C at the rate of 0.5 °C/min. All values are accurate to ( 1 °C. Experiments are repeated at least three times, and all values are the average of three
valid measurements. b For nomenclature, AP-PNA is used for any PNA containing at least one aminopeptoid side chain. The first suffix number in
the name specifies the length (the number of methylene carbons) of the side chain, and the second denotes the number of peptoid modifications in the
PNA. Similarly in the sequence, the superscript number on a residue specifies the length of a peptoid side chain on that residue, and the superscript
letter a denotes the amino headgroup of the peptoid side chain. c 5′d(AGTGATCTAC). d 5′d(CATCTAGTGA). e 5′d(AGTGGTCTAC). f 5′(AGUGAUCUAC.
g 5′(AGUGGUCUAC). h The thermal transition of these strands was not obvious as compared with others, so this value was difficult to determine accurately.
i The decamer AP-PNAs contain a single aminopeptoid side chain at the thymine residue at position 6.
inevitably generate a chiral center, and the two stereoisomers
often exhibit distinct helical structures and binding behaviors
in hybridizing with DNA and RNA sequences.7-10,13 It is
therefore of the utmost importance to preserve the chiral
integrity of such PNA analogues during the synthetic
process.13b Modifying the γ-nitrogen of the aeg backbone
has also been considered.12 However, in an early study by
the Nielsen group, γ-N methylation was found to have a
negative impact on the hybridizing affinity of the modified
PNAs,12a which probably has deterred further attempts to
modify at this position.
In our efforts to develop PNA-based agents, we decided to
reinvestigate modifications at the γ-nitrogen by appending to
it an alkyl side chain carrying a functional headgroup (Figure
1b). We name the side chain a peptoid-like side chain, as it is
analoguous to the NR-side chains in N-substituted oligoglycines
or peptoids.14 Modification on the γ-nitrogen preserves the
achiral nature of PNA and therefore causes no stereochemistry
complications synthetically. Introducing such a side chain may
also bring about some of the beneficial effects observed of a
similar side chain extended from the R- or γ-C. In addition,
the functional headgroup could also serve as a suitable anchor
point to attach various structural moieties of biophysical and
biochemical interest. Furthermore, given the ease in choosing
the length of the peptoid side chain and the nature of the
functional headgroup, the electrosteric effects of such a side
chain can be examined systematically. Interestingly, we find
that the length of the peptoid-like side chain plays a critical
role in determining the hybridization affinity of the modified
PNA.
We devised a convenient synthetic method for the
synthesis of AP-PNA monomers (Supporting Informa-
tion), which was derived from the strategy used by the
Nielsen group for the synthesis of N-methylated PNAs.12a
We chose t-Boc to protect the peptoid side-chain amine
and Fmoc for the backbone 2° amino group from which
the PNA oligomer would elongate (Figure 1c). The PNA
oligomers were synthesized on Rinkamide PEGA resin
using standard solid-phase Fmoc peptide synthesis pro-
tocols. While coupling onto an aegPNA residue was easily
effected using PyBOP as the coupling reagent, coupling
onto an AP-PNA residue was more difficult owing to
the steric hindrance of its backbone secondary amine and
was achieved through the action of HATU for ∼ 6 h. PNA
oligomers containing aminopeptoid side chains were
readily soluble in water, which facilitated their purification
and hybridization studies.
A decamer PNA sequence with mixed pyrimidine and purine
content was chosen in this study to examine duplex formation
with complementary DNA and RNA. The amino termini of all
PNA oligomers were acetylated. To determine whether the
length of the aminopeptoid side chain has any influence on the
thermal stability of the PNA/DNA or PNA/RNA duplex, a first
set of five PNAs containing a single aminopeptoid thymine
residue at the middle of the sequence were synthesized. The
spacer length between the peptoid amino headgroup and the
backbone in the five PNAs is of 2, 3, 4, 5, and 6 methylenes,
respectively. Table 1 presents the hybridization data of these
modified PNAs in comparison with the unmodified aegPNA.
The data clearly show that the length of the aminopeptoid side
chain plays a critical role in determining the thermal stability,
i.e., the melting temperature (Tm), of the duplex formed between
the modified PNA and the DNA. Introducing an aminopeptoid
side chain of 5 or 6 methylene carbons, i.e., an Nγ-aminopentyl
(9) (a) Pokorski, J. K.; Witschi, M. A.; Purnell, B. L.; Appella, D. H.
J. Am. Chem. Soc. 2004, 126, 15067. (b) Govindaraju, T.; Kumar, V. A.;
Ganesh, K. N. J. Am. Chem. Soc. 2005, 127, 4144
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C.; Gil, R. R.; Ly, D. H. J. Am. Chem. Soc. 2006, 128, 10258. (b)
Yamamoto, Y.; Yoshida, J.; Tedeschi, T.; Corradini, R.; Sforza, S.;
Komiyama, M. Nucleic Acids Symp. Ser. 2006, 50, 109. (c) Ishizuka, T.;
Yoshida, J.; Yamamoto, Y.; Sumaoka, J.; Tedeschi, T.; Corradini, R.; Sforza,
S.; Komiyama, M. Nucleic Acids Res. 2008, 36, 1464
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(11) Englund, E. A.; Appella, D. H. Angew. Chem., Int. Ed. 2007, 46,
1414
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(12) (a) Haaima, G.; Rasmussen, H.; Schmidt, G.; Jensen, D. K.; Kastrup,
J. S.; Stafshede, P. W.; Norden, B.; Buchardt, O.; Nielsen, P. E. New
J. Chem. 1999, 23, 833. (b) Cerea, P.; Giannini, C.; Angelo, S. D.; Licandro,
E.; Maiorana, S.; Marchelli, R. Tetrahedron 2007, 63, 4108
(13) (a) Sforza, S.; Galaverna, G.; Dossena, A.; Corradini, R.; Marchelli,
.
R. Chirality 2002, 14, 591. (b) Sforza, S.; Tedeschi, T.; Corradini, R.;
Ciavardelli, D.; Dossena, A.; Marchelli, R. Eur. J. Org. Chem. 2003, 1056
.
(14) Simon, R. J.; Kania, R. S.; Zuckermann, R. N.; Huebner, V. D.;
Jewell, D. A.; Banville, S.; Ng, S.; Wang, L.; Rosenberg, S.; Marlowe,
C. K.; Spellmeyer, D. C.; Tan, R.; Frankel, A. D.; Santi, D. V.; Cohen,
F. E.; Bartlett, P. A. Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 9367.
2330
Org. Lett., Vol. 11, No. 11, 2009

Table 2. Effects of Multiple Aminopeptoid Modifications on the Thermal Stability (Tm, °C) of AP-PNA/DNA and AP-PNA/RNA
Duplexes
a 5′d(AGTGATCTAC). b 5′d(CATCTAGTGA). c 5′d(AGTGGTCTAC). d 5′(AGUGAUCUAC. e 5′(AGUGGUCUAC). f The thermal transition of these
strands was not obvious as compared with others, therefore this value is difficult to determine accurately.
or Nγ-aminohexyl group, maintains the high thermal stability
of the PNA/DNA duplexes. However, inclusion of a shorter
aminopeptoid side chain (n ) 4, 3, or 2) results in a decrease
in thermal stability of the PNA/DNA duplex, and this negative
effect becomes most detrimental with the shortest two-carbon
spacer which causes an 8.4 °C drop in Tm of the AP-PNA/
DNA(ap) duplex. On the binding of the modified PNA to RNA,
there is also a spacer length-dependent effect on the thermal
stabilities of AP-PNA/RNA duplexes, but the effect is less
pronounced, as the negative impact of a short aminopeptoid
side chain is less severe than in the case of AP-PNA/DNA
duplexes (Table 1). Also, like aegPNA and other backbone-
modified PNAs, these aminopeptoid-modified PNAs exhibit a
higher binding affinity to complementary RNA than to the
DNA. Most importantly, these AP-PNAs bind to antiparallel
DNA or RNA containing a single-base mismatch with remark-
ably lower thermal stability than to the fully matched antiparallel
DNA or RNA. This clearly shows that introducing a peptoid
side chain onto aegPNA does not compromise the binding
specificity of the modified PNA and that these AP-PNAs
maintain the ability to discriminate between closely related
sequences. This aminopeptoid modification also preserves the
preference of PNA for binding to the DNA in the antiparallel
orientation, as the AP-PNAs bind to the parallel DNA sequence
with significantly reduced affinity.
Next, PNAs containing multiple aminopeptoid side chains
were prepared to study the cumulative effect of Nγ-aminoalky-
lation. Summarized in Table 2 are the data obtained on
AP-PNAs of the 2-, 3-, 4-, and 6-C side-chain spacer series.
From these data, one can see that although the negative effect
of a short 2- or 3-C aminopeptoid side chain on the DNA-
hybridizing affinity increases with increasing number of modi-
fications the effect is not additive. This finding is similar to
what was observed by the Nielson group on Nγ-methylated
PNAs.12a One should point out that when the cumulative effect
of multiple Nγ-aminoethylation or aminopropylation lowers the
hybridizing affinity of the modified PNAs to a certain level it
also almost abolishes their binding specificity, as seen in the
cases of PNA 9 and PNA 12. For the 6- and 4-C series, only
a very small or no decrease in thermal stability of the AP-PNA/
DNA(ap) duplex is seen when the number of peptoid modifica-
tions is increased from 1 to 4 or even 5. For the binding to the
full-match antiparallel RNA, it is worth noticing that all
members of the 6-C series display a slightly higher affinity (in
Tm) than aegPNA and that their binding affinity is not affected
by the increasing number of Nγ-aminohexyl modifications. With
only a few exceptions (PNA 9 and PNA 12 basically), most of
these AP-PNAs bind to complementary antiparallel DNA and
RNA in a highly sequence-specific manner, as seen from their
lower binding affinities toward the single base-mismatched
DNA or RNA (Table 2).
It is worth pointing out that, despite the negative impact of
a short peptoid side chain on hybridizing affinity, almost all
the AP-PNAs listed in Table 2 still bind to complementary
antiparallel DNA or RNA with a higher affinity than the
corresponding DNA oligonucleotide (Tm of a comparable DNA/
DNA or DNA/RNA duplex is about 33-34 °C15). The
moderate hybridizing affinity of some of the peptoid PNAs,
such as the 4-C series, may have some practical value for
antisense applications and be exploited for designing antisense
agents with optimal binding affinty and kinetics at physiological
temperature.16 Our data suggest that by choosing an appropriate
side-chain length and/or degree of peptoid modification it is
possible to design PNA-based agents with tailored hybridization
strength. One can even envisage using several mixed peptoid
side chains of different spacer lengths to fine-tune the hybridiza-
tion behaviors of PNA. Our data illustrate again the excellent
versatility of PNA’s aeg backbone, which allows it to be easily
modified to suit a variety of applications.
To further examine the effect of side-chain length on thermal
stability, the amino headgroups of four mono-Nγ-aminoalkylated
PNAs (PNA 2, 3, 4, and 6 from Table 1) were modified by an
aminopropionyl or ꢀ-alanyl group, which extended the side
chain by three atoms, to give PNA 20, 21, 22, and 23,
respectively. As seen from Table 3, aminopropionylation
significantly increased the DNA-binding affinity of the
(15) Jensen, K. K.; Orum, H.; Nielsen, P. E.; Norden, B. Biochemistry
1997, 36, 5072.
(16) (a) Malik, R.; Roy, I. Expert Opin. Drug DiscoVery 2008, 3, 1189.
(b) Meier, C.; Engels, J. W. Angew. Chem., Int. Ed. 1992, 31, 1008.
Org. Lett., Vol. 11, No. 11, 2009
2331

Table 3. Effects of Modifying the Peptoid Side-Chain Amine on Hybridization Property
a 5′d(AGTGATCTAC). b 5′d(AGTGGTCTAC). c 5′(AGUGAUCUAC. d 5′(AGUGGUCUAC). e The thermal transition of these strands was not obvious
as compared with others, therefore this value was difficult to determine accurately.
AP-PNAs with a short side chain, AP-PNA 2-1 and
AP-PNA 3-1 notably, but not that of AP-PNA 6-1 which
already has a long side-chain spacer. For example, PNA 20,
whose side-chain spacer is extended from three atoms in
AP-PNA 2-1 to six atoms, gains ∼6 °C in Tm as compared
to AP-PNA 2-1. The fact that PNA 20 is still not as good as
AP-PNA 6-1 despite having a spacer with the same number
of atoms suggests that, in addition to the spacer length, factors
such as intrachain hydrogen-bonding ability and/or conforma-
tional rigidity of the peptoid side chain may also, albeit in a
minor way, affect the binding affinity of AP-PNA toward
complementary oligonucleotides.
a stabilizing effect on the helical structure of the AP-PNA/
DNA duplex, as previous studies on peptoids have shown that
increasing the size of the peptoid side chain can impose
restrictions on the conformational flexibility of the oligoglycine
backbone and be used to induce its helicity.18 However, future
structural investigations by NMR or X-ray crystallography are
needed to find out whether these or any other factors are
involved. The data on PNA 24 and particularly PNA 25 also
confirm that one can indeed use the peptoid side-chain amine
in an AP-PNA as the attachment site to introduce a relatively
large molecular moiety for preparing PNA conjugates with
potential diagnostic and therapeutic interest.
In conclusion, we have shown that the aeg backbone of PNA
can be modified at the γ-nitrogen with a long side chain while
maintaining the high hybridizing affinity and specificity of PNA.
A systematic structure-activity study reveals an interesting
relationship between the hybridizing affinity and the length of
the side chain of the peptoid PNA, which has not been
previously described for other backbone-modified PNA ana-
logues. The amino headgroup in AP-PNAs can be modified
in various ways without affecting the binding affinity, suggesting
that the peptoid side chain can bear a variety of functional
moieties. These findings indicate that modification on the γ-N
of the PNA backbone is capable of generating PNA-based
agents whose hybridization and pharmacological properties can
be modulated, through changing the length and headgroup of
the peptoid side chain, to meet the specific requirements in
different applications.
The above data illustrate again the beneficial effects of a long
side chain in AP-PNA, although the effects seem capped at
the 5- or 6-C spacer length. This observed spacer length-
dependent effect on the hybridizing affinity of AP-PNA is
intriguing. From the data obtained in this study and those of
Nielsen et al. showing the negative impact of Nγ-methylation,
one might be enticed to postulate a favorable role of the positive
charge of the peptoid amine for charge-charge interactions with
the phosphodiester backbone which would compensate for the
negative effect of an Nγ-alkyl group, and a longer 5- or 6-C
spacer would provide the optimal distance for such interactions.
However, acetylation of the positive charge-bearing peptoid
amine of AP-PNA 6-1 causes no reduction in the hybridiza-
tion affinity of the resultant PNA 24 (Table 3). Also, when the
side-chain amine of AP-PNA 6-1 was acylated with biotin,
a useful biochemical probe, the resultant PNA 25 exhibits the
same hybridizing affinity as does AP-PNA 6-1. Therefore,
the positive charge on the amino headgroup of the peptoid side
chain does not seem to play a role in contributing to the
hybridizing affinity of AP-PNA. This observation is also
similar to what was found in a previous study on PNAs with a
γ-C side chain.11 The present data do not allow us to pinpoint
the cause of this length-dependent effect, and it seems that
factors other than charge-charge interactions are at play. One
possible factor is that a short aminopeptoid side chain might
induce a higher content of E configuration about the tertiary
amide due to hydrogen bonding potential of the peptoid
ammonium with the amide carbonyl, whereas PNA’s backbone
amide is known to adopt a Z configuration for oligonucleotide
binding.17 It is also possible that a longer side chain may have
Acknowledgment. The authors thank the Ministry of
Education (MoE) of Singapore for financial support as well as
Nanyang Technological University.
Supporting Information Available: Experimental pro-
1
cedures, H and 13C NMR, HPLC profiles, MS data, and
thermal denaturation curves. This material is available free
of charge via the Internet at http://pubs.acs.org.
OL900587B
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