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LEE D. FADER
YOULA S. TSANTRIZOS
Modifications of Peptide
Nucleic Acid (PNA) monomers
as a strategy for modulating the
physicochemical and pharmacokinetic
properties of PNA oligomers
Tinkering with the flow of genetic information via
sequence selective binding of synthetic oligonucleotides to
DNA, RNA or nucleic acid-binding proteins (e.g.
polymerase or helicase enzymes and transcription
factors), has been an area of intense research activities in
biomedical sciences. The underlying mechanism of gene
silencing, or over-expression, by means of a synthetic
compound is conceptually simple and relies on the
rational, structure-based design of molecules that can
mimic the function of natural oligonucleotides. In principal,
the successful design of such molecules should provide
therapeutic agents and biomedical tools which are
capable of selectively modulating the flow of genetic
information, at either the translation or the transcription
level. In spite of great progress made towards achieving
these goals, the simplicity of the underlying principle
belies many yet unanswered secrets that are utilized by
nature to properly choreograph gene expression.
Recently, a number of review articles have highlighted the
key innovations in the field of oligonucleotide (ODN) and
peptide nucleic acid (PNA) chemistry, as well as
applications of their oligomers in antisense/antigene
technologies, diagnostics and drug discovery efforts1,2.
The focus of this article is to briefly review the key
obstacles hampering progress, and summarize some
recent innovative approaches addressing some of the
problems. The emphasis of the article is on the
contributions of peptide chemistry to the field, as it
pertains to the synthesis of novel PNA oligomers with
modified physicochemical properties.
THERAPEUTIC APPLICATIONS OF
OLIGONUCLEOTIDES (ODN) AND PEPTIDE
NUCLEIC ACID (PNA) OLIGOMERS
Modulation of gene expression with short synthetic
oligomers (ODNs or PNAs) has been achieved by a
variety of mechanisms including, (a) translation arrest
induced by the binding of an antisense oligomer to RNA,
via sequence-specific Watson-Crick base-pairing (Fig. 1,
Pathway a), (b) inhibition of transcription due to the
formation of a triplex between an antigene oligomer and
DNA, via sequence specific Hoogsteen or reversed
Hoogsteen base-pairing (Fig. 1, Pathway b), or (c)
transcription factor decoys which can bind to nuclear
proteins playing a critical role in gene up-/downregulation (Fig. 1, Pathway c).
To date, a significant number of synthetic oligomers have
40
entered clinical development for a variety of therapeutic
targets3. However, only one such molecule (Vitravene™,
ISIS pharmaceuticals) has been commercialized for the
treatment of cytomegalovirus-induced retinitis4. This
relatively poor success rate in the development of ODN
therapeutics is partly due to their (a) synthetic complexity,
(b) poor cell-membrane permeability, (c) lack of efficient
means to deliver the oligomer to specific cells and cellular
compartments (e.g the nucleus), and (d) target specificity.
Although target specificity has been implicated in many
instances, including the toxicity of antisense oligomers
having a phosphorothioate backbone (e.g. Vitravene™),
this issue is of particular relevance in strategies where the
role of the synthetic ODN (or PNA) is to sequester a
DNA-binding transcription factor (Fig. 1, Pathway c).
The multiplicity of target-unrelated genes that are under
the control of a single transcription factor protein has
consequences which are currently difficult to evaluate via
biochemical or phenotypic observations3.
Peptide nucleic acids (PNA, Figure 2) are a unique class
of ODN analogs in that the phosphodiester-deoxyribose
backbone of natural oligonucleotides has been simplified
to an acyclic and achiral polyamide backbone5. This
fascinating class of molecules forms highly sequence
selective and thermodynamically stable complexes with
both DNA and RNA; a finding which has sparked an
immense amount of interest in the development of PNAs
as potential therapeutic agents or biomedical diagnostic
tool6. The potential use of PNA oligomers in antisense
technology has already been demonstrated in vivo7, as
well as in a variety of biomedical applications8.
Nonetheless, PNAs also suffer from many of the same
undesirable pharmaceutical properties that limit the utility
of ODN analogs, including poor cell-membrane
permeability, and lack of delivery control to specific cell
type and cell compartments. In contrast, the notable
advantage of PNA over ODN oligomers is their
dramatically simpler backbone structure, rendering their
large-scale synthesis easily feasible by solution and
solid-phase peptide chemistry.
INNOVATIONS IN PNA STRUCTURE
MODIFICATION TO IMPROVE DRUG DELIVERY
A major issue in pharmaceutical and biomedical
research is the relatively low internalization of PNAs
(as well as ODN) oligomers into target cells9.
Biochemical delivery systems, exploiting the active
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cellular uptake1. Bioconjugate
constructs which modulate the
physical properties of PNAs
have also received significant
attention as a means of
improving bioavailability and
activity
in vivo. For example, selective
upregulation of green
fluorescent protein (EGFP)
expression has been
demonstrated by Sazani and
co-workers in a transgenic
“green mouse”12,13 model
using antisense technology. In
this study, PNAs conjugated
to a cationic lysine
tetrapeptide were found to
exhibit greater in vivo
antisense activity (i.e.
upregulation of EGFP) that
the corresponding uncharged
morpholino or anionic
(2’-O-MOE)-phosphorothioate
oligomers13. However,
chemical modifications of the
PNA structure is a more direct
method for modulating their
Figure 1: Modulation of gene expression via (a) antisense, (b) antigene or (c) transcription factor
biophysical properties.
decoy mechanism; dashed arrows indicate normal flow of genetic information
Given the simplicity of the
PNA structure, large-scale
synthesis of monomer units
can be achieved both
economically and efficiently.
The N-(2-aminoethyl)glycine
backbone is usually
assembled in a single step
from inexpensive
commercially available
starting materials and the
required nucleobase is then
attached in a conventional
peptide coupling reaction
(Scheme 1)14,15. Following a
saponification step, the Nterminal protected free acid
PNA monomers can then be
loaded into a conventional
peptide synthesizer for
Figure 2: Structure of DNA, RNA, PNA and an example of a
oligomer synthesis. Originally, Nielsen and coworkers
modified PNA (GPNA)
optimized each chain elongation step of the solid-phase
synthesis of PNA oligomers and their protocols have been
transport mechanism of key biomolecules, such as
widely employed by others16. Thus, the benefit of any
peptides10, hormones and glycosides11, have led to PNA
bioconjugates with significant organ specificity and
Scheme 1: PNA synthesis
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structural modification must be weighed against the
impact it will have on the synthetic complexity of the new
PNA analogs.
PNAS WITH IMPROVED PHARMACEUTICAL
PROPERTIES - STRUCTURAL MODIFICATIONS
A considerable amount of effort has been devoted to
making structural modifications that could address the
poor aqueous solubility and cell membrane permeability
of the original PNA analog. Early modifications included
replacements of the glycine-derived backbone with other
amino acid residues leading to analogs with improved
hybridization properties17.
Modified synthetic protocols which assured high
enantiomeric integrity of both monomers bearing chiral
residues, such as D-/L-lysine, D-/L-serine, D-glutamic
acid, L-aspartic acid and L-isoleucine, as well as their
corresponding oligomers, were developed18. The choice
of coupling reagent and base used during solid-phase
oligomerarization of chiral PNA monomers were shown
to have a significant impact on the enantiomeric purity of
the product. Surprisingly, extensive HATU-induced
racemization was reported; based on NMR data,
intra-residue deprotonation of the activated PNA
monomer, by either the pyridine or the triazole nitrogen
of the HOAt ester, was proposed as the mechanism of
racemization (Figure 3)19.
Figure 3: Plausible mechanism of HATU-induced racemization
The importance of these studies is clearly evident as an
increasingly high number of chiral PNA analogs have
been found to exhibit improved pharmacokinetic
properties. For example, a
guanidinium-based PNA
oligomer (Figure 2, GPNA)
was designed by Ly and
coworkers20, inspired by the
remarkable cellular uptake of
the human HIV-1 Tat
transduction domain that
comprises short basic
sequences (GRKKRRQRRR).
This PNA analog was shown
to exhibit remarkable
biophysical properties,
including increased solubility
in aqueous media and
significantly higher
hybridization affinity for DNA
as compared to the
unmodified original PNAs.
More importantly, the GPNA
analog exhibited a
dramatically superior uptake
42
and localization into the cell nucleus in cultured HCT116
cells (colon carcinoma cell line), as determined by
fluorescence microscope images20. Furthermore, the fact
that such a simple structural modification of the PNA
backbone could address two of the major drawbacks
limiting applications of PNAs (i.e. solubility and
specificity in intracellular localization) provides strong
validation for the continued interest among chemists in
designing novel PNA analogs which could further
improve the therapeutic utility of this class of compounds.
An additional application of some chiral PNA derivatives
is that their side chains can serve as linkers for the
attachment of biomolecules such as sugars, steroids and
peptides. As previously mentioned, these PNA derivatives
fall under the category of PNA-bioconjugates, which can
take advantage of biochemical delivery systems to enter
cells (i.e. active transporters) and consequently, exhibit
superior pharmaceutical properties.
A considerable amount of effort has also been directed
toward the synthesis of conformationally preorganized
PNA analogs. Notable examples include the NielsenAppella- (8)21, Neislen-Micklefield- (9)22, JordanGanesh- (10)23, Ganesh-Lowe-Liu- (11)24, Lowe- (12)25
and Leumann- type (13)26 analogs shown in Figure 4.
Some of these compounds were designed on the basis of
structural data available from the natural intended targets.
It has been proposed that the introduction of the cyclic
moieties could selectively constrain the dihedral angles
within the polyamide backbone to the desired
conformation, closely
resembling the expected bound
conformation of the PNA-target
complex27. As previously
discussed, a notable feature of
some analogs is the
poly-cationic nature of their
modified backbones, which
confers favorable properties.
In many cases, the introduction
of a cyclic moiety into the
backbone of the PNA increases
the synthetic complexity of the
monomers. However, exceptions to the latter can be
found, such as the synthesis of the pyrrolidinyl analog
1225a. A sub-monomer strategy was employed that
Figure 4: Conformationally preorganized PNA analogs
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Figure 5: Precursor building blocks or the synthesis of pyrrolidinyl analog 1225a.
utilized building blocks 14 and 15 or 16 for the
solid-phase assembly of the oligomers (Fig. 5).
The aminoproline submonomers were prepared from the
corresponding N-nitrosoprolines, which were reduced,
protected and activated using standard procedures.
The substituted proline derivatives 15 or 16, bearing a
nucleobase, were easily accessed from commercially
available trans-hydroxy-D-proline 1724c,28 in 8 steps and
in good overall yield. These building blocks were then
used to prepare oligomers where the average coupling
efficiency was estimated to be >99% (quantified by the
UV absorption of the liberated piperidine-dibenzofulvene
adduct at each deprotection cycle). Hybridization studies
with analog 12 revealed that these PNA derivatives form
highly stable complexes with complementary DNA, and
exhibit higher thermal stability and sequence selectivity
than the original unmodified PNA analogs. Remarkably,
complexes of PNA-12 with RNA were far less stable to
those formed with DNA; this selectivity phenomenon is in
sharp contrast to unmodified PNAs, which generally
exhibit higher binding affinity for RNA than DNA.
These interesting properties, combined with the improved
water solubility imparted by the
poly-cationic backbone, make the pyrrolidinyl analog 12
one of the most interesting PNA derivatives. This example
further demonstrates the potential of
fine-tuning the molecular recognition and biophysical
properties of PNAs by simply modifying the structure of
the monomer units.
Our own contributions to this field have focused on the
introduction of aromatic or heteroaromatic moieties
along the PNA backbone and we have termed these
aromatic peptide nucleic acids (APNAs, Figure 6)14d,29.
The key element in this work is the design of analogs
which can plausibly explore intramolecular π-stacking or
dipole-quadrupole interactions along the oligomer’s
backbone, and between adjacent monomer units, as a
means of preorganizing the APNA oligomer into a
conformation which is favorable for complex formation
preferably with RNA. In an effort to improve water
solubility and conformational pre-organization, a number
of different APNA monomers were synthesized (18-21)
and incorporated into PNA-APNA chimeras and APNA
homopolymers30. Preliminary hybridization studies with
some of these analogs provide some evidence of
beneficial backbone pre-organization. For example,
PNA-APNA chimeras containing multiple, contiguous
APNA units of 20 exhibited far greater hybridization
stability with RNA than the equivalent PNA oligomer
containing only one insert of the APNA monomer 2031.
CONCLUSIONS
The advancement of peptide nucleic acids continues to be
of broad interest in chemistry and biomedical research.
Over the last few years, simple modification of the
original N-(2-aminoethyl)glycine backbone structure
provided further improvement of the already impressive
biological properties of PNAs. Undoubtedly, the next
decade will continue to bring innovative applications of
novel PNAs, PNA-bioconjugates and PNA-ODN chimera
and further close the gap between these remarkable
mimics of natural oligonucleotides and compounds which
can be used as therapeutic agents.
Figure 6: APNA analogs
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LEE D. FADER1
YOULA S. TSANTRIZOS*1,2
* Corresponding author
1. Department of Chemistry, McGill University,
Montreal, Quebec, Canada, H3A 2K6.
2. Department of Chemistry, Boehringer Ingelheim
(Canada) Ltd. Laval (Quebec) Canada, H7S 2G5
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