Peptide Nucleic Acid Conjugates: Synthesis, Properties and

14 Pages·2005·0.12 MB·English

Current Topics in Medicinal Chemistry, 2005, 5, 1119-1131 1119
Peptide Nucleic Acid Conjugates: Synthesis, Properties and Applications
Zhanna V. Zhilina, Amy J. Ziemba and Scot W. Ebbinghaus*
Arizona Cancer Center, University of Arizona, Tucson, 85724, AZ, USA
Abstract: Artificial control of gene expression has great potential in the treatment of many human diseases, and peptide
nucleic acids (PNAs) offer several potential advantages for silencing gene expression in mammalian cells. The
pseudopeptide backbone of the PNA makes it resistant to enzymatic degradation, and PNAs bind complementary DNA
and RNA with high affinity and specificity. PNAs are potentially leading agents for antigene and antisense therapeutics,
but the application of PNAs in the in vivo setting is hampered by their poor intracellular delivery. This problem has been
addressed by PNA conjugation to lipophilic moieties, peptides, and cell-specific receptor ligands. The biological activity
of PNAs can also benefit from conjugation to DNA interactive compounds like intercalators and alkylators. Here we
review the most interesting literature concerning PNA conjugation with small molecules, emphasizing synthetic
approaches, properties and applications of the PNA conjugates.
INTRODUCTION
O
Downregulation of specific gene expression has been a Base NH
goal of chemists and biologists for more than two decades. O Base
Accomplishing this goal could provide treatments for
diseases such as cancer, viral infections, and age-related
N
diseases as well as provide tools for gene knock-down O
O
experiments to dissect the roles of given genes and pathways O P O -
O
in cell biology and disease. Antisense oligonucleotides
O
NH
(ASOs) bind to a complementary RNA and prevent gene
Base Base
expression by RNase degradation or physically blocking O
translation from the target mRNA. Antigene
oligonucleotides (AGOs) bind to DNA with Hoogsteen N
bonds to form a triple helix and prevent gene expression by O O
interfering with transcription factor binding or physically O P O - O
blocking RNA synthesis (for recent reviews, see [1-5]) At
O NH
least one ASO (Vitravene®), achieved FDA approval for
treating cytomegalovirus retinitis, demonstrating that O Base Base
therapeutic oligonucleotides can be effective for the
treatment of disease, achieve regulatory agency approval, N
and can be commercially manufactured and distributed [6]. O
O
Because DNA and RNA are very labile in biological O
O P O -
systems, most pre-clinical and clinical studies of therapeutic
oligonucleotides have employed phosphorothioate backbone
PNA
modifications because they are relatively easy to synthesize DNA
and impart some resistance to nuclease degradation. Fig. (1). Chemical structures of PNA and DNA. The PNA
Unfortunately, these molecules have suboptimal nuclease backbone consists of 2-aminoethyl glycine linkages in place of the
resistance and are responsible for many biologically ribose-phosphate DNA backbone. The nucleobases are attached to
important unintended effects due to interaction with non- the PNA backbone at the glycine nitrogen with a methylene
target biomolecules, especially proteins [7]. carbonyl linker.
Peptide nucleic acids (PNAs) were first reported as
sequence-specific DNA binding compounds by Peter Nielsen PNAs have many desirable qualities as therapeutic
and colleagues in 1991 [8, 9]. PNAs are DNA mimics in oligonucleotides. PNA oligomers can be easily assembled by
which the sugar-phosphate backbone of DNA is replaced by well-established solid phase peptide synthesis protocols and
an uncharged peptide backbone (Fig. 1), and compared to subsequently modified with reporter groups or peptides with
most synthetic DNA mimetics, PNAs have a higher affinity relative ease. They are highly resistant to degradation and
for complementary DNA and RNA sequences [10]. show few interactions with proteins or other biomolecules
[11, 12]. As RNA binding agents, PNAs bind with high
stability to the target mRNA to physically block translation
*Address correspondence to this author at the Arizona Cancer Center, [13]. As DNA binding agents, PNAs are highly resistant to
University of Arizona, 1515 North Campbell Avenue, Tucson, AZ 85724-
displacement by helicases [14, 15]. These properties make
5024; Tel: [phone]; Fax: [phone]; E-mail:
[email protected]
1568-0266/05 $50.00+.00 © 2005 Bentham Science Publishers Ltd.
1120 Current Topics in Medicinal Chemistry, 2005, Vol. 5, No. 12 Zhilina et al.
PNAs attractive to the field of antisense and antigene to DNA is by double duplex strand invasion : invasion and
technology. Watson-Crick binding to both strands of DNA by
pseudocomplementary PNAs (pcPNAs) bearing modified
Pyrimidine-rich PNAs (py-PNAs) can bind to a
nucleobases [20].
polypurine DNA sequence by strand invasion and form a
PNA:DNA:PNA triple helix, displacing the complementary Studies in cell-free in vitro systems demonstrated the
pyrimidine DNA strand (Fig. 2) [9, 16, 17]. The rate of PNA ability of PNAs to specifically inhibit transcription and
binding and strand invasion may be improved by linking the translation. PNAs can prevent transcription by blocking
two single PNA molecules by a flexible linker in a bis-PNA RNA synthesis. The formation of a strand invasion complex
and the inclusion of positively charged residues such as was shown to sterically hinder RNA polymerase elongation
lysine, creating a cationic PNA (cpy-PNA) [18]. The and completely arrest mRNA synthesis [21]. PNAs can also
formation of a pyrimidine motif triple helix requires prevent transcription by blocking transcription factor binding
protonation of cytosines, which generally requires a low pH. to a DNA recognition sequence and preventing the initiation
In order to form Hoogsteen bonds at neutral pH, cytosines of transcription. For example, a PNA targeted to a NF-k B
are generally substituted with pseudoisocytosine, also called binding site prevented transcription by sterically blocking
the J-base (Fig. 3) [19]. Another mechanism of PNA binding NF-k B binding [22]. PNAs prevent translation by blocking
Fig. (2). Schematic representation of the triplex invasion complex formed by PNA oligomers. PNA1 forms a triple helix with dsDNA
(PNA/DNA/DNA) by Hoogsteen bonds (generally in the parallel orientation with respect to the purine strand of the target DNA). PNA2
invades dsDNA and binds to the purine rich DNA strand by Watson-Crick bonds (in the antiparallel orientation), which causes displacement
of the pyrimidine rich DNA strand and formation of a D-loop.
A B
cytosine+ H Pseudoisocytosine
H H N cytosine N H H
N N H N cytosine
N + H
N N H
backbone H O H N N backbone O H O N N
N H
N N
O N
O
Hoogsteen H
N N N Watson-Crick N N N H
guanine
H guanine
H
backbone
C
CH
3
thymine CH
3
O O thymine
N
N H H
N N N
O H
H
N
N O
N N
adenine
Fig. (3). Triplex formation by homopyrimidine PNA oligomers. (A) C+:G:C base triplet. Protonated cytosine in the PNA forms Hoogsteen
hydrogen bonds with the guanine in a G:C base pair. (B) J:G:C base triplet. Hoogsteen bond formation at neutral pH is facilitated by
substitution of the cytosine in the PNA by pseudoisocytosine (J-base), which bears a proton at the N3 position. (C) T:A:T base triplet.
Thymine in the PNA forms Hoogsteen bonds with adenine in an A:T base pair.
Peptide Nucleic Acid Conjugates Current Topics in Medicinal Chemistry, 2005, Vol. 5, No. 12 1121
mRNA processing through the ribosome. Frequently, PNAs [49], electrochemistry [50], and vibrational spectroscopy
are designed to bind at or near the initiation codon, and have [51], and several reports of PNA terminal modifications to
been shown to efficiently inhibit the initiation of translation increase PNA binding affinity have been published [52-54].
[23-25]. PNAs targeting other regions of the mRNA can In this review, we classify PNA conjugates by the type of the
prevent translation. For example, PNAs designed to bind to small molecules which were conjugated to the oligomers,
the Bcl-2 mRNA were shown to prevent translation when overview the synthetic strategies, and summarize the
targeted to the 5’ untranslated region with a complementary properties of the conjugates with respect to the effects on
PNA or when targeted to a polypurine tract in the coding target nucleic acid binding and gene expression in cultured
region with a bis-PNA [26]. The ability of PNAs to bind to a cells.
target mRNA with sufficient stability to prevent translation
GENERAL CONCEPTS FOR MODIFICATION OF
is important, since the PNA:RNA heteroduplex does not
PNAS
activate cellular RNAses. To enhance the antisense activity
of PNAs, RNAse activating agents have been appended to Early PNA synthesis was essentially based on the manual
PNAs, endowing them with the ability to activate RNAse L assembly of monomers by using tert-butyloxycarbonyl (Boc)
[27]. Taken together, these studies show that PNAs can chemistry on solid support [55]. Fluorenylmethoxycarbonyl
effectively and efficiently prevent transcription and (Fmoc) PNA building blocks have now become available,
translation in cell-free systems. and the efficient manual and automatic synthesis of PNA
oligomers by mild Fmoc chemistry is now possible [56, 57].
The poor cellular uptake of PNAs has slowed the
Most PNAs are synthesized on solid support; however,
development of these agents as potential therapeutic
protocols for efficient liquid-phase synthesis of PNAs have
molecules. While the targeting of any class of
also been developed [58]. Internal modification of PNAs has
oligonucleotide to specific cells with subsequent delivery
been reported for the incorporation of linkers, amino acids,
across the cell membrane remains a substantially unsolved
and unnatural nucleobases [19, 59-61]. One method for
problem in the therapeutic setting, many reagents exist to
conjugation to an internal site is provided by the N6-
transport DNA and negatively charged DNA or RNA
aminoalkyl modified adenine (Fig. 4D) [49].
analogs in tissue culture. Like most oligonucleotides, PNAs
are not well transported across the cell membrane in the Different approaches have been developed to create
absence of such reagents, but because most PNAs are PNAs with terminal modifications. PNAs can be conjugated
uncharged or weakly cationic, they cannot be easily used to a small molecule at the N- and C-terminus by using
with most transfection reagents for tissue culture standard or modified protocols for peptide synthesis on solid
experiments [28]. Different delivery approaches for PNAs support or in liquid phase. Most PNA modifications have
have been developed and have been reviewed [29]. been done at the N-terminus, utilizing the free amino group
Microinjection [23], electroporation [30-32], hybridization of the pseudopeptide backbone while the PNA is still
with a partially complementary DNA oligomer for complex attached to the resin and the other reactive groups are
formation with cationic lipids [33, 34], and permeabilization protected (Fig. 4A). The design of PNA N-terminal
of the cell membrane [35, 36] have been used to deliver modification may require a linker between the PNA and the
PNAs into cultured cells to study their biological effects. ligand to position the ligand optimally for interaction with
Another strategy to allow PNAs to traverse the cell DNA or to separate the ligand from the end of the PNA to
membrane involves the attachment of peptides derived from reduce steric interference (Fig. 4B). A 2-aminoethoxy-2-
or related to the cell penetrating peptides (CPPs), and ethoxy acetic acid (AEEA) linker is illustrated, which can be
generally are relatively short and rich in basic amino acids. incorporated into the PNA during solid phase synthesis
Examples used for delivery of PNAs include the retroviral before the addition of the ligand moiety on the solid support.
tat peptide [37], the antennapedia homeodomain (antP) Different polyamide linkers are commercially available that
peptide [38, 39], and the SV40 large T-antigen nuclear can be easily introduced at any position of the PNA oligomer
localization signal (NLS) peptide [40, 41]. Interestingly, (N- and C-terminus, or in between two PNA sequences in
artificial sequences composed of basic amino acids, such as bis-PNAs). Amino acids such as glycine could also serve as
(AAKK) [41] or simply short (four amino acid) polylysine linkers. Moreover, the side chains of the amino acids can be
4
tails [42, 43] can enter cells and affect biological processes used as sites for conjugation. For example, the e -amino
with a target RNA molecule. Using some of these group of lysine may be used for modification of the PNA at
techniques, it has been possible to demonstrate the ability of the C-terminus (as illustrated in Fig. 4C) or internally. For
PNAs to target a specific mRNA and cause antisense effects this purpose, the acid labile monomethoxytrityl (Mmt)
in mammalian cells, and many of these studies have been protecting group is used for the lysine side chain to allow
reviewed [44-46]. The ability of PNAs to bind to DNA in deprotection under mild conditions while keeping the rest of
cells has also been demonstrated by the antigene activity of the protecting groups in place [41]. The application of a
PNAs [reviewed in [47]], as well as by inhibition of DNA highly orthogonal protecting group strategy in combination
replication [48], activation of transcription [32], and site- with chemoselective conjugation reactions can provide
directed mutagenesis [35]. access to rapid and automatic solid phase synthesis of dual
labeled PNAs.
PNA conjugation with small molecules can also promote
the cellular uptake of PNAs, as well as improve the binding CONJUGATION WITH FLUORESCENT MARKERS
efficiency, kinetics, and biological activity of PNAs. Several
PNA-small molecule conjugates have been synthesized to For application as diagnostic probes, PNAs have to be
facilitate detection of PNAs using fluorescence spectroscopy equipped with reporter groups. Labeling techniques utilizing
1122 Current Topics in Medicinal Chemistry, 2005, Vol. 5, No. 12 Zhilina et al.
Base Base
O O
O O
Solid support
N N
H2N NH n NH Spacer A
Base Base
O O
O O O
Linker
H2N O N N
O N NH NH Spacer B
H n
Base Base
O O
O O O
C
N N NH Spacer
Fmoc-HN NH NH
n
(CH2)4
N-e -Mmt-Lysine
NH(CH ) NH-Fmoc NH-Mmt
26
N6-aminoalkyl adenine
N
N
Base N N Base
O O O
O O O
N N N
Boc-HN NH NH NH Spacer D
n
Fig. (4). PNA templates on solid support for conjugation with activated small molecules. The small molecules can be conjugated to the free
amino group at the N-terminus of the pseudopeptide backbone (A) or a linker (B). Modification of the C-terminus can be accomplished by
conjugation with the e-amino group of a lysine initially protected with an acid labile Mmt group (C). Internal modification of a PNA can be
accomplished by introduction of an N6-aminoalkyl adenine into the PNA chain at any position, making the amino group of the adenine
available for conjugation (D).
enzymes are not applicable to PNAs since they are not benzhydryloxycarbonyl (Bhoc) protecting groups on the
substrates for DNA-modifying enzymes. Instead, PNAs have PNA and HYCRON resin enables the application of mild
to be functionalized by chemical methods, and the synthetic cleavage conditions. The removal of all protecting groups
aspects of the conjugation between PNAs and fluorophores would proceed on the solid phase being followed by labeling
have been thoroughly described in the literature [49]. on resin. Use of palladium (Pd0) catalyzed cleavage from
Fluorescent markers are generally appended to the N- HYCRON resin provides mild cleavage conditions that do
terminal amino group of the PNA by reaction with not degrade fluorophores. The choice of resins is important
commercially available probes, such as the isothiocyanate because the resins can affect coupling efficiencies by
derivatives or succinimidyl esters of fluorescein and interacting with the reactants [63]. In particular, polyethylene
rhodamine (Fig. 5) [57, 62-64]. glycol-polystyrene (PEG-PS) resins absorb activated
fluorophores and lead to inefficient labeling of PNAs. That
Attachment of the fluorophore to the C-terminus and to
problem can be solved by adding an inexpensive, inactivate
internal conjugation sites have also been reported [65, 66].
fluorophore to block the resin prior to labeling.
For example, fluorescein-ethylenediamine was conjugated to
the C-terminal carboxyl group of a PNA in solution. The C- Fluorescently labeled PNAs have been used to
terminus is generally inaccessible when the PNA is on solid characterize structural and thermodynamic aspects of the
support, so that a combination of internal and N-terminal PNA/DNA interactions. Fluorescence resonance energy
labeling reactions can be used for on-resin labeling. For transfer (FRET), absorption hypochromicity (ABS),
instance, the N6-aminoalkyl modified adenine was used for isothermal titration calorimetry (ITC), and circular dichroism
efficient conjugation with the fluorescence quencher 4- (CD) techniques were employed to evaluate PNA/PNA and
(4’dimethylaminophenylazo)benzene sulfonic acid chloride PNA/DNA duplex hybridization. Overall, the methods give
(dabsyl chloride). In order to avoid degradation of the consistent values of enthalpy and entropy of hybridization,
fluorescent labels from strong acids used during cleavage but the presence of the fluorophore can affect the results. For
from the solid support, a combination of a highly orthogonal example, fluorophores were shown to stabilize the
blocking group strategy with chemoselective conjugation PNA/PNA duplex; in this instance, fluorescein conjugated
reactions can be used. For example, the use of Fmoc and via a flexible linker to one of the strands in the PNA/PNA
Peptide Nucleic Acid Conjugates Current Topics in Medicinal Chemistry, 2005, Vol. 5, No. 12 1123
HO O O
F F Cl
H C CH
O 3 3
F N CH3 H
OH CH3
O O
O F F Cl CH3
O
N phenylacetate mustard,
pentafluorophenyl ester O H H
O
O O
6-carboxyfluorescein, Me Cl cholesteryl chloroformate
succinimidyl ester
N+ I-
O
O
O
N
O N N
O
4-benzoylbenzoic acid, Me N+ Mn O (CH2)4COOH
succinimidyl ester N N
I-
HN
2
O H
HN N O
OH N+ I- N HN
Me OH
N N
manganese tris (methylpyridiniumyl) Hoechst analog
6-(9-acridinyl)aminohexanoic acid porphyrin carboxylic acid carboxilyc acid
Fig. (5). Example of small molecules functionalized for conjugated with PNAs by solid phase synthesis.
duplex stabilized the right-handed helical form, probably via hybridization (FISH) methods [68-70]. Fluorescently labeled
an electrostatic interaction between the negatively charged PNAs were designed to target specific rRNA sequences of
fluorescein and a lysine side chain on the opposite PNA different microorganisms and evaluated by PNA FISH,
strand [64]. The introduction of fluorescent labels is known allowing the simultaneous identification of multiple species
to affect DNA duplex stability, particularly when attached to in a mixed population, which has never been accomplished
an internal base [67]. Similarly, Seitz and colleagues with DNA probes.
demonstrated that an unlabeled 16-mer PNA forms a duplex
There is little information about the binding of
with a complementary DNA oligonucleotide, yielding
fluorescently labeled PNAs to double stranded DNA. Studies
melting temperatures (T ) that are 6.5-8.5ºC higher than
M conducted in our laboratory demonstrated that a 9-mer cpy-
duplexes with internally or end-labeled PNAs. At the same
PNA modified with 5-carboxytetramethylrhodamine
time, the T of a fluorescently labeled PNA hybridized to a
M (TAMRA) interacts with dsDNA differently than the
complementary DNA oligomer is similar to the T of the
M unmodified 9-mer cpy-PNA. Electrophoretic mobility shift
unmodified DNA/DNA duplex, which can be explained by
analysis (EMSA) showed that the presence of the TAMRA
higher stability of the PNA/DNA duplex compensating for
significantly accelerated DNA binding, while the
the perturbation caused by the fluorophore. The same study
transfection of luciferase reporter plasmids treated with the
showed that single-stranded PNA probes were only weakly
PNA-TAMRA conjugate demonstrated an increase in both
fluorescent but experienced a substantial fluorescence
target and non-target DNA binding by the conjugate,
enhancement when bound to complimentary DNA, which is
measured by a decrease in luciferase activity by a plasmid
useful for studying PNA/DNA interactions [49]. At the same
bearing a PNA binding site as well as a control plasmid
time, it should be noted that the fluorescence properties of
without a PNA binding site. In contrast, the unmodified PNA
the dyes conjugated to PNAs are highly temperature
was unable to bind to plasmid DNA with sufficient stability
dependent, which is important if the fluorescent characteris-
in cells to either the intended target sequence or non-target
tics of labeled PNAs are used for quantitative purposes [64].
DNA to alter the luciferase activity of reporter plasmids.
There is little information about the thermodynamic
Fluorescently labeled PNAs have also been used as tools
binding properties of fluorescently labeled PNAs to
to follow the subcellular trafficking of plasmid DNA.
complementary RNA. At the same time, a potentially useful
Fluorescently labeled PNAs can be bound to plasmids
application of the fluorescently labeled PNAs is the
engineered to contain a PNA binding site [71, 72].
identification of microorganisms by fluorescent in situ
1124 Current Topics in Medicinal Chemistry, 2005, Vol. 5, No. 12 Zhilina et al.
Fluorescently labeled, triplex forming PNAs were used to create DNA probes whose binding affinity does not depend
demonstrate that cationic lipids efficiently delivered the on the purine content of the probe [77]. The stability of the
PNA-complexed plasmids to CV-1 cells, and the plasmids PNA/DNA duplex depends on the purine content of PNA
localized to vesicles within the cytoplasm shortly after strand, which makes the application of PNA probes difficult
transfection [72]. Fluorescently-labeled bis-PNAs were used in mixed sequences [78]. PNAs equipped with a functional
to evaluate the nuclear localization of plasmid DNA, and group to stabilize the PNA/DNA duplex could help to
following microinjection, they were able to follow the overcome that limitation, and a planar aromatic moiety,
movement of the plasmid into the nuclei of individual cells could stabilize the PNA/DNA duplex by p -stacking
in real time [73]. Studies with fluorescently labeled PNAs interactions. Naphthalene diimide was conjugated to the
conducted in our research group demonstrated PNA PNA N-terminus with a polyamide linker and the resulting
localization in the nucleus of cancer cells after delivery by PNA-NADI conjugate demonstrated the expected stabilizing
electroporation. The use of fluorescently labeled PNAs to effect on the DNA/PNA duplex. NADI has geometric
track PNA uptake and subcellular localization in mammalian parameters that closely resemble those of natural AT and GC
cells in culture has been recently reviewed, and in this pairs, and NADI can efficiently interact with DNA duplexes
setting, cell penetrating peptides (CPPs) have frequently and triplexes [79-81]. The authors took advantage of the two
been used to transport PNAs designed as antisense or imide groups of NADI, one of which was modified with
antigene agents into cells [29]. In a recent example, a carboxylic group, and the second with an Alloc protecting
fluorescently labeled antisense PNA conjugated to a group. This construct allowed ligation between the PNA N-
membrane-permeating peptide was designed to target first terminus and the carboxylic group on the functionalized
six codons of the B-cell lymphoma/leukemia-2 (bcl-2) proto- NADI while the PNA was on the solid support. Subsequent
oncogene [74]. Scanning confocal fluorescence microscopy cleavage of the Alloc group under mild conditions will
demonstrated the ability of the conjugate to cross the cell render the second imide accessible for subsequent
membrane and concentrate in the cytoplasm, where high conjugation on solid support, and positively charged
concentrations of bcl-2 mRNA are expected to be present. macrocyclic ligands were conjugated to this imide in order to
improve PNA solubility and binding to negatively charged
PNA-INTERCALATOR CONJUGATES
DNA. A polyamide linker between the PNA and the NADI
Intercalators are a class of small molecules that have of not shorter than 5 atoms in length was needed for the
been used for site-directed DNA modification to alter target NADI to be well-positioned for interaction with DNA. These
gene expression [75]. Nielsen and colleagues first used the studies further demonstrated that the PNA-NADI conjugate
idea of conjugating a PNA to acridine to improve PNA increased the thermal stability of the PNA/DNA duplexes.
strand invasion at elevated ionic strength [54]. High ionic Similar to acridine, PNA modification with NADI did not
strength stabilizes the target DNA duplex and is unfavorable alter PNA sequence specificity. Studies with other PNA-
to strand invasion of the target duplex by the PNA. Mono- intercalator conjugates have demonstrated relatively weak
and bis-PNAs were conjugated to 9-aminoacridine by amide stabilizing or destabilizing effects on the PNA/DNA duplex.
bond formation between the PNA amino terminus and 6-(9- For example, conjugation of a PNA to the dianionic 1,4,5,8-
acridinyl)aminohexanoic acid (Fig. 5) with the PNA on its naphthalenetetracaboxylic moiety resulted in destabilization
solid support [76]. PNA binding studies conducted at various of the PNA/DNA duplex by 12.1(cid:176) C in thermal denaturation
ionic strengths demonstrated that conjugation to acridine studies, data which are in agreement with studies of PNAs
significantly enhanced PNA binding at physiological ionic conjugated to analogously charged fluorescein [64].
strength and improved the kinetics of DNA strand invasion.
PNA-intercalator conjugates may improve the biological
The authors concluded that the acridine, which relies on a
activity of the PNA in antisense applications. For example, a
combination of hydrophobic and electrostatic forces for
PNA-acridine conjugate demonstrated improved antisense
DNA binding and is much less sensitive to ionic strength,
activity against the MDM2 oncogene mRNA in JAR cells,
brings a high local concentration of the PNA in close
reflected in a decrease in levels of the MDM2 protein and a
proximity to the target DNA. The high local concentration of
subsequent increase in p53 levels [82]. Moreover, this
the PNA increases the probability of encountering a
conjugate was cytotoxic to the cancer cells alone and
transiently open DNA double helix in a “breathing event.”
enhanced the activity of the topoisomerase I inhibitor
Significantly, the enhanced DNA binding efficiency by the
camptothecin. PNA-intercalator conjugates could constitute
PNA-acridine conjugate did not compromise binding
a lead for further developments of anti-cancer drugs.
specificity.
PNAS CONJUGATED TO PHOTOREACTIVE
Mokhir and colleagues modified PNAs with a variety of
AGENTS
DNA intercalators that were neutral, positively charged, or
negatively charged [53]. Conjugation of intercalators PNAs have been conjugated to photoreactive agents in an
containing amino groups, such as 1,5-diaminonaphthalene attempt to stabilize PNA binding to DNA or as probes of the
and 3,8-diamino-6-phenyl-phenanthridine, to PNAs was structure of PNA/DNA complexes. Psoralen and benzophe-
accomplished by coupling of a bromoacetyl group to the none derivatives are representatives of the photoreactive
PNA N-terminus using bromoacetyl bromide with a crosslinkers, which upon irradiation with ultraviolet (UV)
subsequent amination reaction between the bromine and the light interact with pyrimidine bases, especially thymine, to
amino group of intercalator. form crosslinks via photocycloaddition reactions [83]. PNAs
appended with these photoreactive molecules could
Derivatives of the neutral dimeric intercalator
potentially form covalent bonds with the DNA target
naphthalene diimide (NADI) was conjugated to PNAs to
Peptide Nucleic Acid Conjugates Current Topics in Medicinal Chemistry, 2005, Vol. 5, No. 12 1125
sequence after UV irradiation, thus stabilizing the binding of Nitrogen mustards are a class of alkylating agent widely
the PNA to the target sequence. The strong fluorescence used as cancer chemotherapies and immunosuppressives [88-
exhibited by these photoreactive molecules can also be used 90]. The biological activity of the nitrogen mustards, like
to monitor PNA interactions with other biomolecules. The most DNA alkylating agents, is mediated through the
literature records several examples of the synthesis and electrophilic attack of the N7 position of guanine bases, and
applications of PNAs conjugated to photoreactive molecules the cytotoxicity of the bifunctional nitrogen mustards is
[52, 84, 85]. Synthetically, PNA-psoralen conjugates were correlated to the formation of DNA interstrand crosslinks.
prepared by coupling 8-methoxypsoralen to the N-terminus The efficiency of the nitrogen mustards is limited by
of the PNA on its solid support using standard Boc protocols deactivation due to hydrolysis and by reaction with
[52]. In a second example, a PNA-benzoylbenzoic acid nucleophilic biomolecules other than DNA. The studies
conjugate was prepared by conjugation of the N- conducted in our research group on triplex forming (DNA)
hydroxysuccinimido ester of 4-benzoylbenzoic acid to the oligonucleotide-nitrogen mustard conjugates demonstrated
PNA N-terminus [84]. that these agents could, in principle, downregulate oncogene
expression in cancer cells [91]; [92]. We postulated that
The ability of these agents to stabilize PNA binding to
PNAs conjugated with nitrogen mustards would be
DNA was shown with melting studies and with denaturing
advantageous because of the nuclease resistance and high
EMSAs. The PNA-psoralen conjugate bound to a
affinity for DNA contributed by the PNA, and that these
complementary DNA sequence with higher stability than an
unmodified PNA, probably due to the p -stacking of the agents could rapidly deliver the alkylating agent to the target
sequence in DNA, thus avoiding deactivation. Unmodified
aromatic rings of psoralen with the flanking base of the DNA
PNAs targeted to a polypurine tract in the HER-2/neu
strand [52]. The interaction of the psoralen with the flanking
promoter were able to suppress HER-2/neu transcription by
base also resulted in quenching the fluorescence intensity of
about 50% in reporter plasmids transfected into tumor cells
the psoralen. Conjugation to psoralen did not reduce PNA
[36], and we expected to observe improved antigene activity
specificity for the target sequence. In this instance, the
by conjugating the PNAs to nitrogen mustards. Furthermore,
stabilization of DNA binding is due to the non-covalent
PNA-alkylator conjugates could represent interesting tools to
stacking interactions of the psoralen with the adjacent
study the mechanisms of double stranded DNA binding by
flanking sequence, but covalent photo-adduct formation with
mono- and bis-cpy-PNAs by positioning the alkylating
the target sequence was not reported. A PNA-benzophenone
moiety at the N- or C- terminus of the PNA and analyzing
conjugate was shown to form covalent photo-adducts with
the sites of DNA alkylation on the target sequence over time.
the target DNA sequence, demonstrating in principle that
this strategy can be used for site-directed covalent bond We synthesized several PNA-alkylator conjugates with
formation between PNA and DNA. Studies comparing the nitrogen mustards phenylacetic acid mustard (PAM) and
binding with a single mismatched target sequence showed chlorambucil (Chl) by solid phase synthesis. The nitrogen
that the PNA-benzophenone conjugate retains selectivity for mustards were activated for conjugation by forming the
the target sequence [84]. pentafluorophenyl (PFP) esters of the PAM or Chl, and these
reagents were conjugated to the PNA N-terminus or to a
Finally, a PNA-anthraquinone conjugate was used to
lysine side chain at the C-terminus. In order to modify the C-
mediate site-specific photo-cleavage to help deduce the
terminus, the PNA was synthesized beginning with a lysine
structure(s) of the complexes formed between the PNA and
bearing a monomethoxytrityl (Mmt) protecting group on the
DNA. The compound was prepared by first conjugating
e -amino. After removal of the Mmt on the e -amino group
anthraquinone-2,3-anhydride to glycine and then adding the
under mild conditions and subsequent reaction with PFP-Chl
glycine-anthraquinone to the PNA by solid phase synthesis
resulted in conjugation only at C-terminus. Interestingly,
using standard conditions for PNA monomer coupling [85].
mono-cpy-PNAs appended with nitrogen mustards stabilized
The strongest cleavage sites were observed in the displaced
the formation of a PNA/DNA/DNA triple helix, and the
strand, probably because the anthraquinone moiety loops
formation of a PNA/DNA/PNA strand invasion complex was
back from the strand invasion complex, rather than
slowed by the presence of the alkylator. In contrast to PNA-
extending along the DNA groove [85]. This work shows the
acridine conjugates, PNA-alkylator conjugates do not appear
potential utility of site-directed photo-cleavage with PNA
to enhance strand invasion. The location of the guanine
conjugates in deducing nucleic acid structure.
adducts formed by the PNA-alkylator conjugates are easily
PNA-ALKYLATOR CONJUGATES detected by inducing strand breaks with piperidine. These
studies revealed not only the PNA binding site, but also the
The interaction between DNA and photosensitive
orientation and order of triplex formation and strand
molecules is a form of photo-induced alkylation, where the
invasion. In contrast to the PNA-anthraquinone conjugate,
excited triplet state of the photosensitive molecule undergoes
the nitrogen mustards most likely extend along the major
cycloaddition to the double bond in the heterocycle of the
groove of the duplex DNA adjacent to the PNA/DNA
pyrimidine (thymine) nucleobase, producing oxetanes [86].
complex and covalently bind to the exposed N7 of guanines
DNA alkylating agents that do not depend on photo-
near the duplex/triplex junction (Fig. 6).
activation have been extensively used in chemotherapy and
would make interesting ligands for conjugation with PNAs Transient transfection of the PNA-nitrogen mustard
[87]; however, no reports of PNA-alkylators have been conjugates bound to a HER-2/neu promoter-luciferase
published so far. construct was used to evaluate the ability of these
compounds to prevent HER-2/neu transcription. In this
assay, a mono-cpy-PNA conjugated to PAM was capable of
1126 Current Topics in Medicinal Chemistry, 2005, Vol. 5, No. 12 Zhilina et al.
Fig. (6). Schematic of a PNA-alkylator conjugate binding to DNA. The mono-cpy-PNA binds to duplex DNA by Hoogsteen bonds (PNA1)
and then binds by strand invasion to form Watson-Crick base pairs with the purine strand of DNA (PNA2), displacing the pyrimidine strand
of the target duplex.
suppressing HER-2/neu expression by approximately 80%, with formation of expected B-DNA like helix, which was
while the unmodified PNA showed less than 20% observed by CD spectroscopy.
downregulation. Moreover, the effect of the PNA-PAM
Unlike the ruthenium complex, a cationic manganese
conjugate on HER-2/neu transcription was observed over 72
porphyrin complex conjugated to the N-terminus of a PNA
hours, while the effect of unmodified PNAs lost after 24
dramatically improved the binding efficiency of the PNA
hours, suggesting that stabilization of the PNA/DNA
[97]. The PNA-metal complex conjugate was synthesized by
complex with the alkylating agent results in more durable
reaction between the carboxyl group in manganese
antigene activity. Such PNA-alkylator conjugates provide a
tris(methylpyridiumyl)porphyrin carboxylic acid (Fig. 5) and
new class of probes for sequence-specific PNA/DNA
the N-terminal amino group of the PNA in liquid phase. The
interaction and might be useful to study gene regulation in
authors ascribed the improved binding efficiency of the
cells.
conjugate to the high affinity of the metalloporphyrin
PNA-METAL COMPLEX DERIVATIVES AND PNA- fragment for the minor groove of DNA, which can increase
MINOR GROOVE BINDER CONJUGATES the local concentration of the PNA and thus increase the
probability of duplex invasion. The metal complexes can
Metal-complexes conjugated to PNA can be used in a
also be used to cleave DNA after the addition of oxidizing
similar fashion to photosensitive molecules to detect PNA
reagents [98]. Oxidation activation of the manganese
binding and to analyze the structure of the PNA/DNA
porphyrin with potassium monopersulfate resulted in
complexes. Metal complexes could provide sensitive
sequence specific cleavage of both DNA strands at the
detection because of their improved fluorescent properties
duplex-triplex junction, indicating that the cleavage took
and could also be used in electrochemical assays [93-95].
place from the minor groove in accordance with the expected
Nolte and colleagues synthesized transition metal derivatives
binding mode of the porphyrin. This study provides another
of thymine PNA monomers, which upon incorporation into
example of how the ability of the PNA to report its binding
the PNA chain would provide the PNA with a unique
site, in this case by oxidative cleavage, can help to study the
spectroscopic handle [51]. PNAs modified with ferrocene
rules of PNA/DNA complex formation and for mapping the
could be used for electrochemical detection, (benzene)-
PNA binding site in DNA. This example also shows how a
chromiumtricarbonyl-PNA would be a sensitive probe for
PNA can serve as an ideal delivery agent to target specific
infrared detection, and ruthenium derivatives of PNAs would
sites in nucleic acids with DNA interactive ligands.
be invaluable tools for the study of electron transfer. These
Nielsen’s group recently reported the synthesis of a non-
new compounds showed unique spectroscopic features,
metal PNA-minor groove binder conjugate [99]. An amino
which in principle permit their sensitive detection even in
acid-Hoechst analogue (Fig. 5) was conjugated to the PNA
complex biological matrices such as intact cells. Encouraged
N-terminus with a 30-atom hydrophilic linker on solid phase.
by these results, the same group reported PNA conjugation
Hybridization with DNA demonstrated that the Hoechst
with 2 transition-metal complexes, namely a ferrocene
ligand increases affinity of the conjugate, analogous to the
derivative and a tris(bipyridine)ruthenium (II) complex [50].
effect of 9-aminoacridine. Moreover, the minor groove
Ferrocene carboxylic acid and the ruthenium complex
binder provides a helps to direct the specific-sequence
(synthesized as described in [96]) were successfully
binding of the PNA and to make PNA binding less sensitive
conjugated to the PNA-N-terminus on solid phase. UV-
to elevation of ionic strength. Furthermore, the PNA-
absorption and melting temperature experiments showed that
Hoechst conjugate demonstrated significantly faster binding
the forward binding kinetics of PNA/DNA duplex formation
to the target sequence than the unmodified PNA, which may
and stability are diminished by the bulky Ru complex,
be explained by the nature of the ligand and the chosen target
possibly by disrupting the hydrogen bonding of the N-
sequence. This observation is interesting in light of work
terminal base pair. The metal complex also affected the
from our group with a triplex forming (DNA)
secondary structure of the PNA/DNA duplex by interfering
oligonucleotide (TFO) conjugated to a pyrrolobenzodia-
Peptide Nucleic Acid Conjugates Current Topics in Medicinal Chemistry, 2005, Vol. 5, No. 12 1127
zepine minor groove binder [100]. We observed that the were treated with a combination of Gd-DOTA-PNA-peptide
minor groove binder slowed the binding of the TFO to the and 111In-DOTA-PNA-peptide, which increased the tumor to
target sequence, which may reflect an inherently slow rate of muscle ratio of radionuclide uptake by 4-fold compared to
binding by the pyrrolobenzodiazepine or the problem of 111In-DOTA-PNA-peptide alone. This study demonstrated
using a major groove binding TFO to direct a minor groove that PDAP dendrimers with 8 DOTA chelators attached to
binder. Because PNAs bind to double stranded DNA by the PNA-peptide conjugate can be selectively taken into and
strand invasion, they have better access to the minor groove, retained within tumors in vivo for scintigraphic imaging. It
and a combination of PNA and minor groove binder appears will be interesting to see whether the molecular basis for the
to improve the binding by the PNA. selective imaging of the tumor cells with these conjugates is
due to greater import by the tumor cells compared to other
Radiometal bioconjugates have been extensively used in
tissues or due to retention of the PNA by binding to the
cancer diagnosis and therapy. Monoclonal antibodies labeled
target mRNA sequence selectively in cells expressing the
with various radioisotopes have been under continued
mutant K-ras mRNA.
development for the imaging and treatment of cancer,
yielding several examples of clinically useful compounds, PNAS CONJUGATED TO LIPOPHILIC MOLECULES
and their evolution has been extensively reviewed [101-103].
Conjugation of PNAs to lipophilic molecules is
The combination of the potential antisense or antigene
motivated by the need to improve PNA transport into the
targeting properties of PNAs with the diagnostic and
cell. Microinjection, electroporation, and permeabilization of
therapeutic properties of the radiometals might create
the cell membrane are useful for in vitro “proof-of-principle”
compounds that could be used in analogous diagnostic and
experiments, but at the expense of significant technical
therapeutic applications.
drawbacks and limited applications for antisense and
One of the approaches for PNA modification with antigene studies. In order to make PNAs effective as
radiometals starts with conjugation to bifunctional antigene or antisense agents, more practical methods of
derivatives of 1,4,7,10-tetraazacyclododecane-N,N,N’,N’’- delivery are needed, which ideally would allow the PNA
tetraacetic acid (DOTA), which can be incorporated into any itself to penetrate the cell. One approach which has been
position of a PNA by standard solid-phase coupling widely applied is the use of peptide carriers for cellular
techniques. DOTA chelates a number of radiometals with delivery [37, 38, 106]. PNAs have also been conjugated to
high stability under physiological conditions, which is ligands for receptor mediated uptake into cells with
essential to avoid excessive radiation damage to non-target molecules such as lactose, mannose, dihydrotestosterone,
cells. Lewis and colleagues reported the synthesis of and insulin like growth factor I [107-110]. Lipophilic
radiometal-labeled PNA-peptide conjugates targeting the molecules that have been conjugated to PNAs to enhance
mRNA of the bcl-2 oncogene [74, 104]. Reaction between their uptake in cells include cholesterol, cholic acid,
DOTA-tris(tert-butyl ester) and the e -amino group of lysine adamantly acetic acid, and triphenylphosphonium. Some of
resulted in a DOTA-lysine intermediate, which was then the other small molecules described in this review are also
attached manually to the N-terminus of the PNA on solid lipophilic (acridine, psoralen, anthraquinone), but none of
phase, followed by the addition of the peptide sequence them were studied in the context of facilitating the uptake of
using automated peptide synthesis. The final construct was PNAs into cells.
labeled with the diagnostic imaging radiometal 111In and the
therapeutic radiometal 90Y. Hybridization experiments Cholesterol and its analogues are highly lipophilic
demonstrated that 90Y-PNA-peptide conjugate was able to molecules which have been used to improve cellular delivery
of anticancer agents [111-113]. Some studies have shown
bind to immobilized bcl-2 mRNA with high sensitivity and
specificity equivalent to a 32P-labeled antisense DNA improved efficacy of cholesterol-conjugated antisense
oligonucleotides and siRNA [114-116]. Our research group
oligonucleotide, and with greater thermodynamic stability
recently modified a PNA with cholesterol by adopting the
[104]. The uptake of fluorescently labeled analogs in cell
synthesis strategy used for polyamines and
culture was reported [74], but no results on imaging or
polyethylenimines [117, 118]. Cholesteryl chlorofomate was
therapeutic applications have been published so far.
conjugated to the PNA N-terminus on solid phase, the PNA-
In a similar approach, Wickstrom and colleagues cholesterol conjugate retains its ability to bind to its target
prepared novel DOTA-polydiaminopropanoate (DOTA- DNA sequence, and evaluation of PNA-cholesterol uptake in
PDAP) dendrimers, which were conjugated to PNA-peptide cultured cells is currently in progress. Similar to cholesterol,
chimeras designed to target the mRNA of the mutant K-ras bile acids are highly lipophilic and have been used in tissue
oncogene [105]. The synthesis started with assembly of the selective drug delivery [119, 120]. Bile acids are the major
delivery peptide on solid phase, followed by inline coupling organic components of the bile, are needed for the absorption
of the PNA monomers. The N-terminus of the PNA moiety of dietary lipids, and are selectively taken into liver and
was then conjugated with different numbers of intestinal cells during enterohepatic recirculation by specific
diaminopropanoate residues, each carrying 2 amino groups membrane transport systems [121]. The substrate specificity
available for conjugation with more diaminopropanoates or of the bile acid transporter has been extensively
with the activated ester of DOTA. The PNA-peptide characterized, and demonstrates a preference for
chimeras were successfully conjugated with up to 16 DOTA trihydroxylated bile acids, such as cholic acid [122]. The
units. Octa-DOTA conjugates were used for labeling with hydroxyl group at the C3 position of bile acids is not needed
111In for scintigraphic imaging or with 157Gd as a contrast for binding to the transporter, and can be used for
reagent for MRI. Mice bearing pancreatic tumor xenografts conjugation. Cholic acid was conjugated to the PNA N-
1128 Current Topics in Medicinal Chemistry, 2005, Vol. 5, No. 12 Zhilina et al.
terminus on solid phase, but free hydroxyl groups in the these results. Stable PNA binding to target nucleic acids
product were overacylated with cholic acid residues [53]. under physiological conditions is also a limitation to the
Melting experiments with the PNA-cholic acid conjugate applicability of PNAs, and conjugation to some DNA
demonstrated that binding to complementary DNA was interactive compounds appear to help and at least partially
weakly improved (D Tm = 3.5(cid:176) C), which is in contrast to improve the biological effects of PNAs. A “modular”
DNA-cholic acid conjugates that have shown a substantial approach to the optimal design of PNAs may be needed,
increase (D Tm = 8-11(cid:176) C) in the thermal stability of DNA where the PNA provides the specificity for binding to the
duplexes [123]. These observations support previous target nucleic acid sequence, and is conjugated to different
findings that ligands with high affinity for the DNA duplex modules that mediate delivery and enhance binding to the
do not necessarily bind well to the PNA-DNA duplex [124]. target sequence under physiologic conditions.
Uptake of the PNA-cholic acid conjugate by the bile acid
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