Peptide Nucleic Acids (PNAs), A Chemical Overview

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Peptide Nucleic Acids (PNAs), A Chemical Overview Andrea Porcheddu* and Giampaolo Giacomelli Dipartimento di Chimica via Vienna 2, Sassari 07100 Italy Abstract: Peptide nucleic acid (PNA) is a nucleic acid analogue and a fully synthetic DNA/RNA-recognising ligand with a neutral peptide-like backbone. In spite of the large change on the backbone structure, PNA molecules bind strongly to complementary DNA and RNA sequences. Originally conceived as ligand for the recognition of double stranded DNA, the unique physico-chemical properties of PNAs have led to the development of a variety of research and diagnostic assays. The extraordinary properties of PNA may advance routine clinical tests and environmental analyses that will utilise the PNA technology. PNAs may also have an impact on in situ hybridisation, cytogenetics and industrial microbiology. This paper presents some recent achievements on peptide nucleic acids and discusses, from the viewpoint of literature, what the potential is and what the limitations of such compounds are. This review, which is not intended to be exhaustive, is mostly aimed at the current progress in PNA chemistry, structure, and hybridisation, highlighting some applications too.

Keywords: PNA, peptide nucleic acid, DNA, RNA, oligonucleotide, duplex, triplex, purine, pyrimidine, PCR. 1. INTRODUCTION Nowadays, there is significant interest in the discovery and development of small molecules that target doublestranded DNA (dsDNA) in a site-selective manner but without serious sequence limitations. Such ligands may yield new robust biomolecular tools, sensitive DNA diagnostics and potent gene therapeutics. For these purposes, dsDNA-ligand complexes have to be sufficiently stable to compete with DNA-processing proteins. The resistance of a ligand to enzymatic degradation is also often essential to allow efficient DNA manipulation. Therefore, ligands are required, which bind dsDNA with (i) high affinity and (ii) high selectivity, also characterized by (iii) sufficient biostability and (iv) lack of sequence restrictions. Despite the fact that many natural and nonnatural (synthetic and semi-synthetic) dsDNA-binding ligands with groove-specific or intercalative modes of complex formation have been identified [1, 2], none of them meets all of these four key requirements simultaneously. Therefore, the search for new ligands with alternative modes of binding to DNA duplexes is of paramount importance. Peptide nucleic acids (PNAs) are nuclease/protease resistant dsDNA-binding ligands that are capable of forming very stable and highly sequence-specific helix invasion complexes with target sites on duplex DNA. The study of peptide-based nucleic acid analogues takes its origin from nearly thirty years ago [3-5]. Jones and co-workers prepared PNA 1 from amino acids with nucleobases attached at the [β -position of alanine [6, 7] (Fig. 1 ). However, no interaction was observed between the PNA 1 and polyadenylic acid. Molecular modelling by Weller et al. predicted that PNA 2 might hybridise to the complementary nucleic acids [8]. But no successful result has been reported. These results

suggest that either the peptide main chain and side chain may be too rigid or steric unfits may prevent to achieve the hybridisation. To increase the side chain flexibility, a single methylene unit as PNA 3 elongated the spacer between the main chain and the nucleobase. The latter oligopeptides containing γ -substituted homoalanines have been synthesised [9], but no hybridisation has been reported. Similarly, PNA 4 that carries BCH2CH2- side groups at the amide nitrogen did not hybridise to DNAs [10]. These results suggest that PNAs that consist of a conventional polypeptide backbone with B-CH2 - or B-CH2 -CH 2 - side groups do not hybridise to DNAs, because of insufficient flexibility of the polypeptide chain. To relax the rigidity of the polypeptide backbone, an ester linkage was introduced as the depsipeptide PNA 5 [8]. Unfortunately, however, the ester bond was easily hydrolyzed in aqueous media. PNA 6 originated from efforts during the 1980s in organic chemist Prof. Ole Buchardt’s laboratory in Copenhagen together with biochemist Peter Nielsen to develop new nucleic acid sequence-specific reagents. In 1991, Nielsen and co-workers discovered that PNAs of δamino acids with a [NH-CH2 -CH 2 -N(CO-CH 2 -B)-CH 2 C O ] n ,-type structure [PNA 6] hybridise to the complementary DNAs with higher affinity than the DNADNA counterpart [11]. Today’s PNAs (Fig. 1) are DNA analogues in which a 2aminoethyl-glycine linkage generally replaces the normal phosphodiester backbone (PNA 6) [12]. A methyl carbonyl linker connects natural as well as unusual nucleotide bases to this backbone at the amino nitrogens. PNAs are non-ionic, achiral molecules and are not susceptible to hydrolytic (enzymatic) cleavage. Despite all these variations from natural nucleic acids, PNA is still capable of sequencespecific binding to DNA as well as RNA obeying the Watson-Crick hydrogen bonding rules [13, 14]. Its hybrid complexes exhibit extraordinary thermal stability and display unique ionic strength properties.

*Address correspondence to this author at the Dipartimento di Chimica via Vienna 2, Sassari 07100 Italy; E-mail: [email protected] 0929-8673/05 $50.00+.00

© 2005 Bentham Science Publishers Ltd.

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B

B B N H

O

B N H

R N

O N H

O

n

PNA 1

PNA 2

O

n

PNA 3

O

O O

B

O O

O N

N H

n

n

PNA 4

B

O

N

N H

O

n

B

N H

O

O

H N

O n

O P O

n

DNA

PNA 5

PNA 6

B = Nucleobas e

Fig. (1). Polypeptide backbones in various PNAs.

amino terminus of PNA is facing the 3’ end of the DNA) with a T m [18] of 50.6 °C [13]. Interestingly, PNA is also able to bind to a parallel DNA targets (i.e. the amino terminus of PNA facing the 5’-end of the DNA), although with lower affinity (56.1 °C). Kinetic binding studies have shown that the binding of PNA to anti-parallel DNA is much faster (< 30 s) than compared to parallel targets [19]. The influence of single-point mismatches is more

Properties of PNA What made the discovery of PNA invaluable is that, although being an artificial nucleic acid, PNA was binding DNA and especially RNA with an affinity greater than that of native nucleic acids [15-17]. For instance, a pentadecamer H-TGTACGTCACAACTA-NH2 was forming a duplex (Fig 2A) with a complementary anti- parallel DNA (i.e. the

B

A

DNA D Hoogsten Watson-Crick

C

DNA Loop PNA

PNA

R N

E

O N

Hoogsten binding

H

O

T

N N

N R

N

O

H N

A

R

N

O HN

H

N C

R

R

G H

N O

NH H

N

H 2N

H

N C+ N

T

O

R

N

NH H

N NH

O Watson-Crick binding

Fig. (2). Schema of PNA binding modes for targeting double stranded DNA. PNA oligomers are drawn in bold. (A) Standard duplex invasion complex formed with some homopurine PNAs. (B) Double-duplex invasion complex, very stable but only possible with PNAs containing modified nucleobases. (C ) Conventional triple helical structure (triplex) formed with cytosine-rich homopyrimidine PNAs binding to complementary homopurine DNA targets. (D) Stable triplex invasion complex, leading to the displacement of the second DNA strand into a “D-loop”. (E) H-bonding in nucleobases leading to Watson-Crick and Hoogsteen complementary. R = ribose or deoxyribose.

Peptide Nucleic Acids (PNAs)

pronounced in PNA-DNA than DNA-DNA duplex. For example, a single Cyt to Gua mismatch in the middle of a DNA pentadecamer showed a Tm depression of 9 °C when hybridised to DNA, but 16 °C in the case of PNA [12]. The increased stability of PNA-DNA and PNA-RNA duplex in comparison to DNA-DNA (RNA) duplex is mainly attributed to the lack of electrostatic repulsion between the two strands. This is supported by experiments showing that the thermal melting stability of DNA-DNA duplex increases with increasing ionic strength above 1 M NaCl. At the same time, the change in ionic strength has little effect on the stability of PNA-DNA duplexes [12]. Homopyrimidine PNAs or PNAs with a high pyrimidine/purine ratio bind to complementary DNA via the formation of (PNA)2-DNA triplexes [12] (Fig. 2C). These complexes are very stable and are dependent on the length of the oligomers; an average increase of 10°C per base pair is observed [19]. Triplex formation involving Cyt is pH dependent, in accordance to the Hoogsteen bind model, i.e. cytosine needs to be protonated at N3 in order to form a hydrogen bond to the N7 of guanine (Fig. 2E). Thermal melting of (PNA)2 -DNA hybrids exhibits pronounced hysteresis, i.e. the difference between the melting (higher) and annealing temperatures (lower), indicating that the rate of formation of the triplex is very slow [12, 20]. Homopyrimidine PNA oligomers, when targeting dsDNA, displaces the pyrimidine strand of the dsDNA and binds to the purine strand forming a (PNA)2-DNA triplex and a looped out ssDNA so called P-loop (Fig. 2D) [21-23]. This process is unique for PNA and takes place only in a low ionic strength buffer (< 50 mM). However, once preformed in low salt buffer, the P-loop structures are stable in salt concentrations as high as 500 mM [24, 25]. The invention of the PNA has launched the development of new approaches in this area. The use of PNA-based probes makes it now possible to more effectively target internally located dsDNA sites. Recently, Demidov et al. [26] have proposed another approach for binding a probe to dsDNA, which is based on the PNA-assisted assembly of a stable complex between dsDNA and an oligonucleotide or other probes with mixed purine-pyrimidine sequence in a proteinfree system. The idea proved to be very fruitful and laid the foundation of PD-loop technology [27], which employs the formation of so-called PD-loops (i.e., the PNA-distended DNA loops) [28] and related structures [29]. Advantages and Disadvantages of PNA Compared to Native Nucleic Acids PNA has several advantages over oligodeoxy- and oligoribonucleotides [30]. •

•

PNA is generally more chemically stable than DNA or RNA fragments. Being a polyamide-based molecule, PNA is very stable under acid and moderately stable under basic conditions, as well elevated temperatures. PNA is also biochemically stable: it is not a substrate for proteases, peptidases or nucleases. These two

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features (chemical and biochemical stabilities) facilitate synthesis, purification, storage and application of PNA oligomers. •

The lack of charges in the PNA backbone results in the lack of electrostatic repulsion between the hybridising PNA-DNA (RNA) strands, and thus in a greater affinity towards its targets. Actually, introduction of a positive charge in the PNA strand could be beneficial for the formation and the stability of duplex and triple helices.

•

PNA molecules are also stable over a wide pH range.

•

PNA is a more sequence-specific binder; single-point mismatches are better discriminated by PNA than by DNA or RNA.

•

PNA forms stable triplexes with DNA, while the (DNA)3 hybrids under certain circumstances may be rather unstable.

•

PNA binding to its complementary single-stranded targets is rather unaffected by the ionic strength of the medium.

PNA has also Some Shortcomings •

PNA has lower solubility compared to both DNA and RNA due to the lack of charge in the backbone; solubility enhancers can alleviate this problem [31].

•

PNA solubility is also related to the length of the oligomer and purine/pyrimidine ratio [32].

•

PNA has very low cellular permeability, thus limiting its applications for antigene or antisense therapies [33-35].

•

Neutral PNA molecules have a tendency to aggregate to a degree that is dependent on the sequence of the oligomer [15].

•

In biological system, the triplex formation is limited only to guanine-poor targets, since the physiological pH does not affect the protonation of cytidine residues [36].

2. PNA SYNTHESIS A ubiquitous requirement in the field of peptide nucleic acid (PNA) research [37, 30] is the preparation of monomers for subsequent oligomerisation. A PNA monomer consists of N-protected (2-aminoethyl)glycine (Pg1) to which a protected nucleobase (Pg2) is attached (Fig. 3). These two protecting groups have to be orthogonal, i.e. Pg2 must be stable to the conditions used to remove Pg1 [38]. Base(P g2) O Pg1

N H

O N O

Pg 3

aeg -PNA monomer

Fig. (3). General structures of a PNA monomer.

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There are several combinations of protecting groups reported for the PNA synthesis, and the most commonly used are summarised in Table 1.

In the next step, protected nucleobases are attached via an amide bond using a coupling agent, e.g. DCC [14] or HBTU [42] (Fig 5).

Table 1.

Although the monomers, suitable for both Boc and Fmoc strategies of solid-phase peptide synthesis, are commercially available, they are expensive and of limited variety. For researchers developing unusual or modified monomers, there is usually the necessity to prepare N-(2aminoethyl)glycine derivatives. Driven by the need to prepare PNA monomers, Hudson et al. have developed a reliable, convenient, and scalable route to ethyl N-(2-Bocaminoethyl)glycinate (1) [52] without explicit purification (Fig. 6). Hudson’s procedure is based on the formation of the imine (5) from N-Boc-ethylendiamine (4) and ethyl glyoxylate hydrate (3), itself obtained by oxidative cleavage of diethyl tartrate (2), followed by reduction of the imine (5) to afford the desired secondary amine (1) without the possibility of over alkylation. A key factor in the success of this scheme is the method by which ethyl glyoxylate is prepared.

Commonly used Protecting Groups Strategies for PNA Synthesis

Entry

Pg1

Pg2

Removal of Pg1

Removal of Pg2

References

1

Boc

Cbz

50% TFA

HF or TfOH

[12, 39-41]

2

Fmoc

Bhoc

20% piperidine

95% TFA

[42]

3

MMT

Acyl

2% DCA

NH3

[43-46]

4

Fmoc

MMT

20% piperidine

2% DCA

[47]

Boc/Cbz and Fmoc/Bhoc PNA monomers are commercially available [48] and are used for routine PNA synthesis with the possibility of using commercial peptide synthesisers. Synthesis of PNA Monomers Although PNA monomers are commercially available, they are also relatively easy to synthesise from inexpensive starting materials. The synthesis of backbone units can be Pg1

Pg1

O N H

O

H 2N

+

To demonstrate the scalability of the procedure (1) has been prepared on 2-, 10-, and 38-g scales [52]. On each scale, the desired product was isolated pure and in essentially quantitative yield. For scale-up, the amounts of reagents and solvents were scaled equally, and reaction times

Pg3

Na(CN)BH3 O

O NH2

N H

+

O

Br

Pg

Et 3N Pg3

H N

N H

Pg3

O

O

Fig (4). Synthesis of protected N-(2-aminoethyl)glycine.

achieved either via reductive amination of glycine ester with an N-protected amino acetaldehyde [49] or via an alkylation

remained the same. The only difference between the scales was the method of hydrogenation.

Base (Pg 2) O

OH +

O Pg1

Base(Pg2)

Pg3 O

HN

O O

DCC or HBTU Pg1

NH

N H

N

O Aeg-PNA monomer

Pg 3

Fig. (5). Synthesis of Aeg-PNA monomer.

of mono-protected ethylenediamine with a bromoacetic ester [50, 51]. Both routes give high yields of the desired intermediate (Fig. 4). OH

OH

O NaIO4,

EtO

OEt O

EtO O

OH 2

BocHN OH

CH2Cl2/H2O

The commonly used methods for the preparation of PNA monomers require several steps to generate the N -(2aminoethyl)glycine unit followed by N-acylation of the

BocHN

5

OEt

CH2Cl2

BocHN

Base

Base H N

O

O

OH

OEt DCC/HOBt 1 N-(2-Boc-aminoethyl)gly cinate

Fig. (6). Synthesis of ethyl N-[(2-Boc-amino)-ethyl]glycinate 1.

OEt

5

3 O

H2, 10% Pd/C

N

BocHN

3 A° M S, CH2Cl2

O N

O

NH2 (4)

BocHN

N

O OEt

Peptide Nucleic Acids (PNAs)

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Base O

Base + RNH2 6 COOH 7

1

+ R CHO 8

C

+

N

NHPg

O

(1) UGI-4CC

9

R

N

N H 10

R1 Base + R2CHO + C N COOH

O N

R

Base

Base Base

O

N H R'

NH2

(3) UGI-4CC

R

R

O N R1

N H 13

12

repeat (3), (4) NH2

N H

R2

R

N H

2

R

O

O

N

N H

R1

Base O

O

N

(4) deprotection

NHPg

Base O

O

N

R

N H

1

O

N

Bas e O

O

O

O

N

11

Base

Base

O

NHPg

(2) deprotection NHPg

N

N H

R2

n

R3

O N H

NH2

PNA Oligomer

Fig. (7). Synthetic scheme of PNA monomer and chain extension; B = nucleobase, Pg = protecting group.

glycine derivative by a carboxymethylated nucleobase. Several quite different research reports have demonstrated the usefulness of Ugi four-component reaction (Ugi-4CR) in the synthesis of PNA monomers [53].

may be selected as four types of building blocks to construct derivatised PNA libraries by solid- or solution-phase synthesis following this strategy.

More recently, Xu et al. have described a new method [54] to obtain PNA monomer and PNA chain prolongation by progressional Ugi-4CR reactions, which differs from classical peptide chemistry in that prior preparation of PNA monomers is not needed as they are built up along with the extended chain.

Solid Phase Synthesis

In this work, PNA oligomer was designed by a repeated protocol of two steps (an Ugi reaction and a deprotection) in one turn (Fig. 7). In the first cycle, a nucleobase-acetic acid (6), an amine (7), an oxo compound [aldehyde or ketone, (8)], and an N-protected aminoethyl isocyanide (9) were used as starting materials in the Ugi-4CR reaction to generate the amino-protected PNA monomer (10) in a one-pot process, followed by deprotection of the amino group. Then the deprotected product (11) reacts as the amine component with the other three components in the second cycle of Ugi reaction to yield the amino-protected PNA dimer (12). By repeating the Ugi reaction and amino-deprotection, the PNA chain can be extended. This strategy can be applied in the synthesis of a number of structural derivatives and analogueues of PNA by employment of different reactants, (6), (7), (8) and (9). More expediently, the four components

The assembly of PNA is usually performed on a solid support, preferably via automated synthesis [55, 56]. The solid support is usually functionalised polystyrene to which a N-protected amino acid (e.g. glycine) is attached via its Cterminus to cleavable linker (Fig 8). It is not advisable to link the PNA monomers directly onto the linker owing to the potential formation of ketopiperazine (14) during the first deprotection step; the support may get de-functionalised [42]. The choice of the linker is very important and depends on the protecting group strategy used. For instance, a Wang linker [4-(hydroxymethyl)phenol is used for Fmoc/Bhoc chemistry and the product is cleaved from the resin by acid treatment, for instance using TFA [57], while a more acid stable HMBA [4-(hydroxymethyl)benzoic acid] linker is used for MMT/Acyl chemistry [46, 58]. The loading of the solid support can range from 30 mM/g to >1 mM/g. Low loading resin is usually used in the synthesis of PNA-DNA chimera [63]. The PNA synthesis cycle consists of the following operations: 1) chain elongation (coupling step), 2)

Bas e O

O

O

N H2N

Linker O

O HN

Linker +

N Bas e 14

Fig. (8). De-functionalisation of support due to cyclisation.

HO

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wash, 3) removal of the transient protecting group and 4) final wash.

As a solution to this problem, Hudson et al. have envisioned a sub monomer synthesis of PNA oligomers (Fig. 9) [65]. On the basis of the Fukuyama-Mitsunobu alkylation, a resin-bound amino acid is first sulfonylated and then alkylated under Mitsunobu conditions with an Nprotected 2-aminoethanol and then desulfonylated. The secondary amino group (15) is then acylated with the desired protected nucleobase-acetic acid derivative, yielding the resin-bound PNA monomer (16). At this point, rather than cleaving and purifying the monomer for use in an oligomer synthesis, the next monomer (17) is constructed on the first one by deprotection of the amino group (16), coupling of another amino acid, which is deprotected, and repetition of the cycle (Fig. 9). Subsequently, oligomerisation may be achieved by deprotection, coupling of another amino acid, and repetition of the cycle.

The chain elongation is the critical step in the PNA assembly. The efficiency of the elongation step, and thus the choice of the coupling agent is crucial for the overall yield and purity of the desired PNA, especially when somewhat longer fragments are to be assembled. HATU is probably the most commonly used coupling agent for the PNA synthesis. It is reported to give better than 99% coupling yields in a relatively short time (15-30 min) [59]. Usually, the sizes of the PNA fragments that are constructed on a solid-support do not exceed 16 bases. There are two main reasons for this: first, a 16-mer PNA has usually already high affinity to both DNA and RNA, greatly surpassing the affinities of the native nucleic acids; second, being rather lipophilic, PNA have a tendency to selfaggregate both on the solid support during synthesis, leading to low yield of the desired product as well as in solution leading to precipitation and non sequence-specific interactions [60, 61]. After synthesis, the PNA is usually purified by reversed-phase HPLC, using conditions similar to those utilised in the purification of small peptides i.e. low pH buffer [62].

3. MODIFIED PNAs PNA-DNA Chimeras Although PNA oligomers have excellent binding properties, they show poor solubility and a tendency to selfaggregation [66]. Preparing DNA-PNA chimeras has solved some of these problems. The type of the linkage at the DNA-PNA junction and the orientation of the PNA tract (NC vs. C-N) in the chimera seem to be crucial for the effectiveness of the recognition processes. These chimeras are more water-soluble, maintain the ability of DNA to activate RNase H and retain some of the enhanced binding and nuclease resistance properties of PNA [67, 68]. There are no electrostatic interstrand repulsions when PNAs hybridise with nucleic acids, and this leads to higher thermodynamic stability than structurally analogueous DNA-DNA or RNADNA. In addition, in contrast to DNA-DNA hybrids, they are stable under low salt condition and it has been shown that PNA and DNA strands have a faster rate of binding relative to DNA-DNA strands. It is possible that PNA-DNA chimeric oligomers in which both types of monomeric units

There are however a few drawbacks which complicate the direct use of PNA in many applications. These problems are most frequently addressed by the conjugation of various moieties to the end of a PNA strand. An alternative to this would be to construct a PNA with an N-(2-aminoethyl)X backbone, where X, rather than glycine, could be any readily available α -amino acid (Fig. 9). Such modifications can impart chirality, charge, or functionality, all without disturbing the bond configuration required for the desired binding properties. The construction of such oligomers first requires the synthesis of the appropriate PNA monomers. This can either be done by reductive alkylation [63] or Fukuyama-Mitsunobu alkylation [64] of the desired αamino acid, followed by acylation with the desired nucleobase-acetic acid derivative. These processes can be tedious and time-consuming.

Base O

O

O NH2

X

H N

a X

R1

R

X

o-Ns

O

H N

b,c

d

NHDMT

resin-bo und amino acid

N

X

R1

1

O

R1

15

NHDMT 16

e Base

PNA oligomers

O

a, b, c

Base O

N

X R1

17

O

O

O NH2

N H

f, g

N

X R2

Reagents and conditions: (a) o-NsCl, DIEA, CH2Cl2; (b) DMTNHCH2CH2OH, TMAD, PBu3, DIEA, THF; (c) KSPh, NMP, H2O; (d) BaseCH2COF, DIEA, DMAP, NMP; (e) TFA, MeOH, CH2Cl2; (f) Fmoc-amino acid, peptide coupling; (g) Piperidine, DMF, X = O or NH

Fig. (9). Submonomer synthesis cycle based on Fukuyama-mitsunobu amine synthesis.

R1

NH3

Peptide Nucleic Acids (PNAs)

Current Medicinal Chemistry, 2005, Vol. 12, No. 22

O 2N

O

O NH N H

O

O

Br

Br

NH

a

NH

O

18

N

O

N H

NH

d N

O

N

O t

21

COOH

O

e

O Bu

N

b

O

O

22

O

Br

2567

Ot Bu

NH O N

f

O O O N

HN

19

CN

O

N

c

O i

N

O

N

O t

O Bu

24

Ot Bu O

O

O

O

23

NH

NHBzl

NHBzl N

N

g

h O

25

Br

N

N

MMT

NH2

NHBzl

NHBzl

O

N

N O O HN MMT

N

N O

NHEt3

O

20 HN MMT

N

N

j

O

O O

O O

CN

26

HN MMT

N

O O

27 NHEt3

Reagents and conditions: (a) Bro moacetic acid, NaOH;(b) N-(2-MMT-amino)ethyl)Glycine 2-cyanoethy l esther, DIP, HOBt, N-Ethylmorpholine; (c) DBU; (d ) t-Butyl bromoacetate, DBU; (e) 2-mesytylenesulfonyl chloride, DMAP followed by o-nitrophenol; 1,4-diazabicyclo[2.2.2]octane; (f) ammon ia; (g) Benzoyl Chloride, pyridine;(h) TFA; (i) N-(2 -MMT-aminoethyl)glycine 2-cyanoethyl ester, DIP, HOBt, N-ethylmorpholine; (j) DBU.

Fig. (10). Preparation of the PNA monomer of 5-bromouracil 20 and 5-methylcytosine 27.

are present in a single chain might combine the favourable hybridisation characteristics of PNA with the high water stability of DNA. Moreover, the structure of such molecules, if it bears a resemblance to DNA, might be compatible with some enzyme-catalysed reactions. Such compounds could therefore be of use in PCR, DNA sequencing, antisense inhibition studies and other technologies. Several authors have described the preparation of DNAPNA chimeras [69]. The most common strategy is to use the MMT group for the temporary protection of the backbone amino function, and acyl groups for the exocyclic amino functions of the nucleobases. The MMT is removed under mild conditions (3% TCA in DCM), and the nucleobaseprotecting groups are removed using concentrated aqueous ammonia. These conditions are similar to those used in DNA synthesis allowing the preparation of PNA-DNA chimeras. The substitution of cytosine by 5-methylcytosine in DNA-PNA chimeras increased duplex stability, while substitution of thymine by 5-bromouracil maintained it (Fig. 10). The PNA monomer carrying 5-bromouracil (20)

and 5-methylcytosine (27) could be obtained following the Eritja’s strategy illustrated in Fig. (10) [70]. Substitution of thymine by 5-bromouracil and cytosine by 5-methylcytosine in DNA-PNA chimeras induced a significant decrease in the stability of triplex indicated by lowering the melting temperatures (17-21 °C for chimeras 29-31 versus 24 °C for chimera 28, Table 2). In contrast, the same substitutions are reported to induce a strong stabilisation of triple helices in oligodeoxynucleotides, indicating that the structure of the triple helices formed by DNA-PNA chimeras are different from the triple helices formed by all-DNA oligomers and for this reason, the mechanisms for their stabilisation are also different. The results obtained with DNA-PNA chimeras carrying 5-bromouracil and 5-methylcytosine are negative, thus precluding their use for triple helix formation. However, the results on triple helix formation obtained with DNA-PNA chimeras are encouraging, since it has

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shown that DNA-PNA chimeras bind to duplex DNA better than PNA oligomers.

In an effort to develop a simpler and more versatile approach to DNA-PNA chimeras, Piccialli et al. [71] have focused their attention on the possibility of using commercially available PNA monomers (35-38), routinely employed for PNA oligomer assembly, as building blocks in an automated synthetic process combining standard phosphoramidite and solid phase chemistry (Fig. 11). In such monomer, the Fmoc group has been introduced to protect the 2-ethylamino moiety in the growing PNA backbone, while the benzhydroxycarbonyl (Bhoc) group was chosen as the protecting group for the exocyclic amino function of A, G, and C bases.

Although PNA oligomers can be designed to form a stable PNA-PNA-DNA triplex via strand-displacement, this process is slow and direct binding to the target duplex DNA may be faster. Table 2.

Melting Temperatures (T m ) of the Triplex Containing DNA-PNA Chimeras (100 mM NaCl, 100 mM sodium phosphate/citric acid pH 5.5). PNA part in italics. M= 5-methyl-C; B= 5-bromo-U and T=T-hydroxyethylglycine as PNADNA linker.

Compound

Sequence (5’- > 3’, N- > C)

Tm (°C)

DNA

CTTCCTCCTCTa

38b

Chimera 28

CTTCCTCCTCTa,c

24

Chimera 29

MTTMMTCCTCTa,c

18

Chimera 30

CTTCCTMMTMTa,c

17

Chimera 31

MTTMMTMMTMTa,c

21

Chimera 32

CTTCCTCCBCBa,c

NT d

PNA

Gly-CTTCCTCCTCTa,c

NT d

Since the Bhoc protecting group was not completely stable to the DCA (dichloroacetic acid) treatments required by the DNA chain assembly procedure, a simple and convenient deprotection/reprotection approach has been developed, involving the removal of such protecting groups on the assembled PNA tract, followed by peracetylation or perbenzoylation of the exocyclic amino functions of the bases. Recently, Mayol et al. [72] have explored the capability of PNA-DNA chimeras to form quadruplex structures. They have reported the usage of 5-phosphoramidite nucleosides for easy preparation of PNA-DNA chimeras containing 5phosphoramidate bonds (Fig. 12). Results from NMR and CD clearly indicate that chimeras 5’TGGG3’-t 50 and t5’GGGT3’ 52 adopt quadruple helical structures. The CD spectra of 50 and 52 show bands characteristic of

aHairpin duplex 5’GAAGGAGGAGATTTTTCTCCTCCTTC 3’. bT =43 °C, at 1M m NaCl, 100 mM sodium phosphate/citric acid pH 5.5. c Terminal 6(hydroxyhexyl)carboxamide. dNT = no transition.

B1 O R1 HN

i)

O ii) Coupling with PNA units 35-38

O

Fmoc

N H

N

O

i) Fmoc removal ii) Coupling with PNA units 35-38

O N H

O

33 R 1 = Fmoc 34 R 1 = H

B1

B1 O

B1 O Fmoc

N H

N

N

HN

O

O O

N

N H

N H

Fmoc

0-2

OH

iv)

DMTO O v) DNA synthesis vi) Detachment and deprotection

O ECO

41 a-e

O

39 B 1 = T, C(Bhoc), A(B hoc), G(Bhoc) 40 B 2 = T, C(Bz), A(B z), G(Bz)

iii)

35 B1 = T 37 B 1 = A (Bhoc) 36 B1 = C(Bhoc) 38 B 1 = G (Bhoc)

5'-DNA-3'p-(N)-PNA-(C) 41 a-f

O

O

B B2 O

N O H

O

O N

P

B2

B = T, A(Bz)

N H

N

O O N H 0-2

O

v i)

Reagents and Conditions: (i) Piperidine/DMF (1:4, v/v, 3x10 min, rt); (ii) (a) 3-6 (8 equiv .), DIPEA (12 equiv.), DIPEA (12 eq uiv.) in DMF, 1 h, rt, (b) Ac 2O/pyperidine (2: 3, v/v, 1 h, rt); (iii) (a) 75% TFA in DCM (w/w), 1h, rt, (b) Ac 2O/pyridine (2: 3, v/v, 1 h, rt) or BzCl/Pyridine (3 :7, v/v, 6h, rt); (iv) as (i), (b) complete coupling cycle with nucleoside-p hos phoroamide (2x20 min, rt); (v) DNA chain assembly on the automated synthesizer; (vi) conc. NH4OH, 16 or 20 h, 5 5°C. CE = 2-cyanoethyl.

Fig. (11). Solid phase synthesis of DNA-3’-PNA chimeras by using Bhoc/Fmoc PNA monomers.

Peptide Nucleic Acids (PNAs)

Current Medicinal Chemistry, 2005, Vol. 12, No. 22

2569

B1 O

O H N O

Fmoc

R

42 R = Fmoc 43 R = H

i

O T

O

O

N

N H

46

O

O

O

O O

O N

N H

N H

51

P O

t-5'GGGT3' 52

ODMT OCE

O N

N H

Fmoc

N H

i, vii

47 B = G(Bhoc) 48 B = G(Bz)

i, vi

ix, x

O

O

B

B O

N

N H T

O

O

O

5'TGGG3'-t 50

OCE

T

i, viii

i, iv, iii

N H

49

Fmoc

ix, x

P

N

N H

O

i, vii

O O

O

O T

N H

O

O ii, iii

ODM T

B O

N OH N H (Bhoc) 44 B = T; 45 B = G

B

T O

O

O

i, viii

O

O N

N

N H

ODMT

B O

N H

53

ODMT

ix, x

O P OCE N H O

ix, x

5'TGG3'-gt 54

O T O O O

N H

B

O

B

O N

N H

O

O N 55

O N H

P O

OCE

tg-5'GGT3 56

Reagents and Conditions: (i) piperidine/DMF, 1:4 v/v; (ii) 44, HATU, DIPEA, DMF/CH3CN, 1:1 v/v; (iii) Ac2O/piperidine, 2:3 v/v (capping step); (iv ) 45, HATU, DIPEA, DMF/CH3CN, 1:1 v/v; (v ) TFA in DCM 75% w/w; (vi) BzCl/pyridine, 3:7 v/v; (vii) coupling with 3 pho sphoramidite monomer; (viii) coupling with 5 -phosph oramidite monomer; (ix) DNA chain assembly on the automated synthesis er; (x) concd. NH4OH.

Fig. (12). Solid phase synthesis of PNA-DNA chimeras: were therefore prepared: 5’ TGGG3’-t (50), 5’TGG3’–gt (52), t-5’GGGT3’ (54) and tg-5’GGT3’ (56).

quadruplexes and are similar to that of [d(TGGGT)]4, which exists as a tetramolecular system. In particular, NMR spectra show that the modified quadruplexes possess fourfold symmetry with all strands parallel to each other and all DNA nucleosides in anti conformations. Furthermore, as indicated by UV experiments, the presence of the PNA moiety at the edge of the quadruplexes, as well as the different PNA-DNA junctions namely (C)-PNA-(N)-p-3’-DNA-5’ (50) and (C)PNA-(N)-p-5’-DNA-3’(52), do not affect the thermal stability of the molecules. On the other hand, the 5’TGG3’gt (54) and tg-5’GGT3’ (56) sequences do not form welldefined structures.

unfavourable on increasing the number of PNA units in the DNA-PNA strands. However, it is interesting to note that PNA-DNA chimeras with only one PNA unit at the 3-ends are still 25 times more stable in human serum than the corresponding unmodified oligodeoxynucleotides [73].

This is probably due to the flexibility of the PNA backbone, which renders quadruplex formation entropically

Since the discovery of peptide nucleic acids as DNA mimics in the early 1990s, a tremendous effort has been

Therefore, substitution of a DNA residue with a PNA one in biologically active quadruplex-forming oligonucleotides (i.e. aptamers) could, in principle, improve the activity of those molecules. PNA-Peptide

2570 Current Medicinal Chemistry, 2005, Vol. 12, No. 22

Porcheddu and Giacomelli

Fmoc

H N 57

Fmoc

i

Rink

H N

H N Rink

(CH2) 4

(v)-(vi) HN

H 2N

Dde

NH2

Peptide

(ii)-(iv)

(CH2)4

(CH2)4 HN

O

O

O

PNA O

O

O Fmoc-SK(Aloc)-NH

Fmoc-GK(Aloc)-NH

Fmoc-YK(Aloc)-NH

NH2

NH2 (CH2)4 NH

NH2 (CH2) 4

(CH2) 4 58

NH Dde-TAC 60

NH Dde-CTT 59

Dde-ACC O H N

Fmoc-GKLS-K(Aloc)

O n

NH2 (CH2)4

61

KPA

Bu

H N

N H

NH2

5 O

O

(CH2) 4

NH

NH

Dde-CTTCCAAAATAC

Dde-CTTCCAAAATAC

62

Reagents and Conditions: (i) NH2OH.HCl/imidazole; (ii) Dde-PNA-OH (5.5 equiv ), PyBop (5 equiv), NEM (11 equiv) in DMF (0.1 M), 3 h; (iii) 20% piperidine in DMF; (iv) Fmoc-aa-OH (5.5 equiv), PyBop(5 equiv ), DIPEA (11 equiv), HOBt (5.5 equiv) in DMF (0.1 M) 3 h; (v) Repeat steps i-iv. (vi) TFA/TIS/CH2Cl2 (90/5/5).

Fig. (13). PNA-peptide conjugate synthesis.

deprotection conditions that were completely orthogonal to Fmoc and apply these to the synthesis of branched PNApeptide conjugates (Fig. 13). They reasoned that this should be possible given the different mechanisms of deprotection for both protecting groups. While Fmoc is cleaved by basic elimination, Dde is cleaved by nucleophiles (trans enamination) [79, 80]. A set of new deprotection conditions and reagents were evaluated using Fmoc-Lys(Dde)-Rink-PS resin (5 7 ) as a simple model compound. Following deprotection, amino groups were coupled with Fmoc-GlyOH as an analytical tag. Bradley et al. have shown that hydroxylamine is the reagent of choice for mild and orthogonal deprotection of the Dde group. This was demonstrated by a highly flexible synthesis of PNA-peptide conjugates (58-62).

directed to their application as antisense and antigene probes [74-76]. With the aim of further enhancing their properties, PNAs have been conjugated to a variety of effector molecules. Among these, small peptide fragments [77], often derived from functional proteins, are able to convey their specific properties to the conjugate. In this context, a highly flexible strategy for the synthesis of branched PNA-peptide conjugates is required. To use commercially available Fmoc-protected amino acid monomers carrying acid-labile protecting groups, PNA monomers with an N-terminal protecting group completely orthogonal to Fmoc are required. The aim of Bradley’s group [78] was to develop novel Dde (1-(4,4-dimethyl-2, 6-dioxacyclohexylidene)ethyl) 63. H2N-TCCCAGGCTCAGATCT-CONH2

NH2 HO

Ring II O Ring I

HO NH2 O 64. R- = *

H N

6N H

NH2 O O

TCCCAGGCTCAGATCT-CONH2

O NH2

HO

R

O 65. Neamine R = H HO

NH2

O 66. Neomycin R- =

HO

O

HO NH2

O

OH

Fig. (14). Structure of the anti-TAR PNA 63, its neamine conjugate 64 and of the aminoglycosides neamine 65 and neomycin 66.

Peptide Nucleic Acids (PNAs)

Current Medicinal Chemistry, 2005, Vol. 12, No. 22

2571

NHTr

NHTr Et3NH O

PMBO

TrHN

PMBO

O

TrHN

O

O O O

OPMB

66

NHTr

H N

NHTr

O

TrHN

PMBO TrHN

a

O

H2N

O

PMBO O

OPMB 67

O NH3

b

4 CF 3CO2 OH

c, b

O

HO

H3N

HO H3N

O

O

O

64

NH3

H N O

Protected PNA

OH

68

O

(a) Succinic anhydride, Et3N, CH2Cl2; (b) TFA/anis ole (1/1); (c) EDC, HOBt, DM F, protected PNA

Fig. (15). Synthesis and deprotection of the Neamine derivative 67 which was conjugated to the protected PNA 63. The conditions are milder than hydrazine and compatible with a larger number of functional groups and solid supports. Furthermore, they have demonstrated that Dde can be employed as an N-terminal group for PNA synthesis. Indeed, this is also the first report of the synthesis of PNA on PEGA resin. Neamine (65) is a constituent of aminoglycoside antibiotics such as neomycin B (66) that are hydrophilic pseudo oligosaccharides possessing several amino functions that are mostly protonated under physiological conditions (Fig. 14). Due to their polycationic nature, they show binding affinity for nucleic acids and bind specifically to 16S bacterial ribosomal RNA (rRNA) and perturb protein synthesis [81]. Pandey et al. [82] have reported for the first time the synthesis and antiviral efficacy of a PNA-neamine conjugate (64) wherein the neamine moiety (65) not only allows cellular uptake of the PNA (63) targeted to the TAR region of the HIV-1 genome but also confers a RNAcleaving activity that may enhance its utility as an antiviral agent. Since the TAR element in the LTR is essential for transactivation of HIV-1 transcription and conserved in all HIV-1 isolates, targeting this invariant region may be an interesting strategy for developing new antiviral agents against drug resistant HIV variants [83, 84]. The polycationic neamine moiety imparts greater solubility to the PNA and also confers a unique RNA cleavage property

Ala

Gly

Cys

Lys

Asn

Phe

to the conjugate that is specific to its target site and functional at physiological concentrations of Mg2+. To link the neamine core (65) to PNA sequences (64) at the 5-position, trityl and 4-methoxybenzyl groups were chosen to protect the amino and hydroxyl functions (66), respectively (Fig. 15). These groups can be easily removed under the conditions used to remove the benzhydryloxycarbonyl (Bhoc) groups employed in the PNA synthesis to protect the amino functions of the bases. The protected neamine derivative (67) was synthesised in six steps (Fig 15). Systemic toxicity is one of the major problems in chemotherapy. While the traditional chemotherapeutics generally affect all of the proliferating cells, new therapy modalities, in particular gene therapy, offer the possibility to selectively act in cancer cells. However, the high molecular weight of the active substances used in these treatment methods leads to unfavourable pharmacokinetic behaviour that causes problems in the clinical application of these substances. Therefore, the success of these treatment methods depends on the development of new vector systems that enable transport of high molecular weight substances for example, oligonucleotides [85, 86]. Somatostatin-receptors (SSTRs) are found in numerous kinds of tumours (e.g., breast tumours, small-cell lung cancer). As the native peptide somatostatin is rapidly

Phe

D Phe

PNA Trp

Cys

Phe D Trp

S S

S S Lys A

Cys

Ser

Thr

Phe

Thr

Lys B

Thr

Cys

Thr

Fig. (16). Comparison of native somatostatin (A) with a conjugate consisting of a PNA and Tyr3 -octreotate (B) The amino acids that are essential for receptor binding are marked in bold.

2572 Current Medicinal Chemistry, 2005, Vol. 12, No. 22

Porcheddu and Giacomelli

O CH2

O

NH

Thr i

Boc

Bzl

O CH2

O

Thr Bzl

ii O CH2

O

Cys

Thr

Lys

D-Trp

Acm Bzl 2-Cl-Z

Phe

For

NH

Boc

S

Cys

Bzl

D-Phe

Acm

S Thr

Cys

Thr

Lys

D-Trp

Acm Bzl 2-Cl-Z

Phe

Cys

D-Phe

NH

Bo c

For

iii S

O CH2

O

Thr Bzl

Cys

S Thr

Lys

D-Trp

Acm Bzl 2-Cl-Z

Phe

Cys

D-Phe

For

CCC

TAC

CGC

GTG

CGA

Tyr

Z ZZ

ZZ Z

ZZ Z

Z ZZ

ZZ Z

2-Br-Z

NH Boc

iv O HO

S Thr

Cys

S Thr

Lys

D-Trp

Phe

Cys

CCC

D-Phe

TAC

CGC

GTG

CGA

Tyr

H

Reagent and conditions: i) Synthesis cycles with HATU/DIEA/Boc cleavage with 5% p-cresol in TFA; ii) Tl(TFA) 3 ; iii) synthesis cy cles; iv) TFA/p-cresol/TFM SA. Protecting groups: Acm = acetamidomethyl, 2-Br-Z = 2-bromobenzyloxycarbonyl, 2-Cl-Z = 2-chlorobenzyloxycarbonyl, Bzl = benzyl, For = formyl, Z=benzyloxycarbonyl, TFA = trifluoro acetic acid, TFMSA = trifluoromethanesulfonic acid, DIPEA = N,Ndiisopropylethylamine,HATU = O-(7-azabenzotriazol-1-yl)-N,N,N’,N’-tetrameth yluronium hexafluorophosphate.

Fig (17). Flow diagram of the synthesis of the PNA–octreotate conjugate using Boc chemistry.

of PNA–peptide conjugates [89]. Octreotate, which is nearly identical to octreotide except that it has a carboxy group at the C terminus and thus supports internalisation (Fig. 16), was chosen as the peptide part of the conjugate.

degraded in vivo, stabilised derivatives have been developed for clinical application. Since, up to now, it has not been possible to selectively transport antisense oligonucleotides into tumour cells, Mier et al. [87] have checked the use of SSTR-affine peptides as carriers for oligonucleotides (Fig. 16).

Boc-d-Phe-Cys(Acm)-Phe-d-Trp-Lys-Thr-Cys(Acm)-ThrPAM resin was synthesised by application of a modified in situ neutralisation protocol [90] with HATU as activator (Fig. 17).

The oligonucleotide target sequence chosen was the antibcl-2-sequence [88]. The convergence of PNA and peptide synthesis enables the stepwise synthesis of PNA–peptide conjugates on the same polymeric support. To allow a convergent synthesis, the peptide part had to be built up by Boc chemistry as well. The PNA oligomers can be synthesised by using Boc chemistry in good yields; consequently, this method is also suitable for the synthesis

To enable the introduction of radioactive iodine (125I), an additional tyrosine residue was conjugated to the N terminus, which led to the target molecule (69) (Fig.18) [91]. In contrast to the phosphorothioate conjugate for the 125Ilabelled PNA–peptide conjugate, a selective enrichment in

Base O

O H 2N

N H

O

N

O

H N

N H n

O

O

H N

O

N H

HO O OH

O

S S

HO

H N

N H

69

Fig. (18). Target molecule 69 (Base sequence= Tyr-AGCGTGCGCCATCCC-peptide).

NH HN

H N O OH

O NH2

Peptide Nucleic Acids (PNAs)

Current Medicinal Chemistry, 2005, Vol. 12, No. 22

the tumour tissue was obtained. The modification of the PNA with the peptide part led to an about ten-fold increase in tumour uptake. The use of SSTR-affine carrier molecules for the first time enables the targeted transport of oligonucleotides in tumour tissue. The introduction of disulfide bridges into peptides usually allows the creation of conformational constraints that can improve the recognition between a ligand and its receptor, therefore improving biological activity [92]. W i c k s t r o m et al. [93] have intended to utilise a disulfide-cyclised D-peptide IGF1 analogue, D(CysSerLysCys) [94], to concentrate oncogene PNA probes in transformed cells that over express IGF1R (insulin-like growth factor receptor) [95], and a cyclic peptide L(CysAsnGlyArgCys) that binds selectively to human breast cancer xenografts in nude mice (Fig.19 and Fig 20). Base O

Peptide1

Spacer1

N H

O N

N H

Spacer2

Peptide2 S

S

12

Fig. (19). Design of trifunctional peptide-PNA-peptides.

To visualise externally those cancerous tissues that take up the PNA-peptide probes and concentrate them by hybridisation to oncogene mRNA targets, they designed trifunctional peptide-PNA-peptides to extend from the Nterminus of the PNA a tetrapeptide, Gly-D-Ala-Gly-Gly, that chelates 99m Tc firmly and efficiently for scintigraphic imaging of γ-particles emitted by decay of 99mTc [96, 97]. PNA-peptide conjugates are usually prepared by fragment condensation [98], which requires multiple steps of

preparation and purification, causing significant loss in yield. Wickstrom hypothesised that continuous solid-phase synthesis on a single-resin support could be extended to Fmoc coupling of all peptide1, PNA, and peptide2 residues during a single run, with Cys-Cys cyclisation on a solid phase, yielding a chimera capable of radionuclide chelation for imaging of gene expression in vivo after a single purification step (Fig. 20). Automated, continuous solidphase synthesis of peptide1-PNA-peptide2 was successful only with a long coupling cycle for the amino acid residues. Chelator peptides were extended from the N-terminus of peptide nucleic acid (PNA) dodecamers, which in turn were extended from the N-termini of disulfide-bridged peptide ligand analogueues, using Fmoc coupling [99] for all residues (Fig. 20). 4-Aminobutyric acid (Aba) was instead selected as a spacer [100] to minimise steric hindrance between the chelator and the PNA. Acetylation of the terminal amino group is a key step prior to obtain disulfide cyclised PNA-peptide chimeras in high purity and high yield when using EtO spacers. The cysteine thiols were cyclised on a solid support, either before or after PNA extension (Fig 20). The melting temperatures of PNA-RNA duplexes implied that the two peptide moieties at the N and C termini of the 12-mer PNA stabilise the duplexes slightly whether the peptides are positively charged or not. 4. PNA BACKBONE MODIFICATIONS Being of a relatively simple structure, PNA monomers are good targets for chemical modifications. By PNA modifications one usually understands changes in the N-(2aminoethyl)glycine backbone and the methylene carbonyl linkage from the backbone to the nucleobase. The scientific

Strategie A S

Acm

Strategie B S

Acm

S Acm S

Fmoc-NH-(Gly) 4-Cys-Asn-Gly-Arg-Cys-CO O Aba =HN

S

S

I 2, DMF, rt, 4h S

PNA Synthesis S

Acm

S

Gly-D-Ala-Gly-Gly-Aba-PNA-(Gly)4-[Cys-X-Y-Cys]-CO

Gly-D-Ala-Gly-Gly-Aba-PNA-(Gly)4-Cys-Asn-Gly-Arg-Cys-CO

TFA, m-cresol, Et3SiH, DCM

TFA, m-cres ol, Et3SiH, DCM

S

Gly-D-Ala-Gly-Gly-Aba-PNA-(Gly) 4-Cys-Asn-Gly-Arg-Cys-CO-NH2

S

Gly-D-Ala-Gly-Gly-Aba-PNA-(Gly) 4-[Cys-X-Y-Cys]-CO-NH2

S Bas e O Peptide1 Sp acer1 N H

Acm S

Gly-D-Ala-Gly-Gly-Aba-PNA-(Gly) 4-[Cys-X-Y-Cys]-CO

Fmoc-NH-(Gly) 4-Cys-As n-Gly-Arg-Cys-CO

S

PAL-PEG-PS

PNA ass embly

S

S

Acm

Fmoc-NH-(Gly) 4-[Cys-X-Y-Cys]-CO

PAL-PEG-PS

I 2, DMF, rt, 4h

2573

O N

Spacer2 Pep tide2 N H S S 12

Fig (20). Automated, continuous solid-phase synthesis of peptide1-PNA-peptide2.

2574 Current Medicinal Chemistry, 2005, Vol. 12, No. 22

Porcheddu and Giacomelli

literature has an ample range of examples reporting PNA modifications [129, 101]. The aim of these modifications has been mostly to further improve the properties of PNA, such as binding affinity and solubility, and to synthesise new DNA mimics in order to get a better understanding of the structural and biological features of the native nucleic acids.

glutamic acid) were shown to destabilise PNA-DNA duplexes further, probably because of the repulsive interactions with the DNA negatively charged phosphate groups [110]. On the other hand, by introducing positively charged lysine-based monomers, it was possible to obtain more stable PNA-DNA duplexes. As far as the configuration of the chiral monomer is concerned, PNAs containing D-monomers (D-PNAs) bind to the complementary antiparallel DNA with greater stability than do the corresponding L-PNAs [110]. The observed enantioselectivity had previously been explained [111] in terms of preferential PNA helicity induced by the configuration of the stereogenic centre. In particular, the Dmonomers were supposed to induce a preferred righthandedness and the L-monomers a preferred left-handedness in the PNA strand: as a consequence, DNA, which is righthanded, would bind preferentially with the right-handed DPNAs. This interpretation was indirectly confirmed by circular dichroism studies of the complexes between canine dyes and PNA-PNA and PNA-DNA duplexes [112].

Chiral PNAs In order to improve the binding specificity, solubility and uptake into cells, the PNA backbone has been modified in many ways. One particular approach involves the introduction of stereogenic centres [102-107], most commonly based on different chiral aminoethylamino acids, first introduced and studied by Nielsen et al. [108]. Chiral PNAs were generally found to form slightly less stable PNA-DNA duplexes than their achiral analogueues [108], the effect being more pronounced for backbones containing amino acids with bulky apolar side chains (such as those derived from phenylalanine or leucine) [109]. Negatively charged monomers (derived from aspartic and

Moreover, with D-Lys-PNAs, it was observed that it was also possible to affect the direction selectivity, i.e. the

O

Cbz

HN NH

N

NH O

N

N H

N

O

O

N

OH

Boc

O

O

N

N H

Boc

OH

N

N H

Cbz-Cl

70b

HN

T-monomer

OH

(H2C) 4

(H2C) 4

HN

N O

O

(H2C) 4 70a

N

O

N

Boc

Cbz

HN

HN

70c

Cbz-Cl

C-monomer

Cbz-Cl

A-monomer Base

H N

Boc

i, ii, iii

HN

H2N

OAllyl

HN O

NH2

N H

N

NH O O N

N

N O

O N H

N

N

N O

O N H

N

N H

NH2N

N O

O N H

NH

N N O

N (CH2)4 NH3

N H

N

O OO N

(CH2) 4 NH3

N H

v

Base = C, 70b Base = A, 70c

NH2 N

O

Base = T, 70a OAllyl

HN

N

O

N

(H2C) 4

NH2 NH

N

iv

72

O

N

Boc

Cbz-Cl

Cbz-Cl

N NH2 N OO N

N H

(H2C)4

NH

N

H N

71

O N

Boc

OH

(H2C) 4

O

O

O

73 a-c Cbz-Cl

O OO N

(CH2) 4 NH3

N H

N O N

NH

NH

N

N

O

NH2

NH2 N

O O

N O N H

N

N

O O O

O N H

N

NH2

D-Lys-PNA 74

i) CH2=CHCH2Br, Cs2CO3, DMF, r. t.; ii) pTSA, toluene, 100 °C; iii) Boc-Gly-H, NaCNBH3, CH3COOH, CH3OH, 0 °C; iv) Base-CH2COOH, DCC, DHBtOH, r.t.; v) morpholine, [Pd(PPh3)4],THF, room temp.

Fig. (21). D-Lys-based PNA monomers containing thymine (70a), cytosine (70b) and adenine (70c) (above) and their synthesis (below).

Peptide Nucleic Acids (PNAs)

With the aim of achieving a selective antiparallel binding with DNA, Marchelli et al. [113] have reported the synthesis and binding abilities of a chiral PNA decamer (7 4 , H G T A G A T C A C T - N H 2 ) bearing three D-Lys-based monomer (a ‘‘chiral box’’) in the middle of the strand (Fig. 21). The antiparallel PNA-DNA duplex showed a melting point of 43 °C (determined both by CD and UV spectroscopy), whereas the parallel PNA-DNA duplex failed to form, as shown by the absence of temperature dependence in the UV and CD spectra. The chiral D-Lys-PNA (74), based on three adjacent Dlysine monomers (‘‘chiral box’’) in the middle of the strand, binds to DNA in exclusively antiparallel fashion, showing no affinity for the parallel DNA target. This is in marked contrast to achiral PNA. Thus, this type of PNA overcomes the lack of binding selectivity in the direction control. Moreover, the same PNA also displayed very good single point mismatch recognition. Both effects seem to arise from a delicate balance between stabilising and destabilising factors. The correct D configuration (entropy effect) and the presence of positive charges (enthalpy effect) favour the duplex formation, whereas steric effects induce destabilisation. Hence, in the antiparallel matched PNA-DNA duplex, good binding still occurs, whereas when new destabilising factors intervene (parallel or mismatched duplexes), it is prevented. Modifications on the Central Tertiary Amide in the Backbone The tertiary amide moiety of the PNA monomer unit that tethers the nucleic acid base to the backbone has received much attention as a target for these modifications. Early molecular modelling investigations suggested that the carbonyl oxygen of the tertiary amide might participate in an intramolecular hydrogen bond, helping to preorganise PNA oligomers for duplex formation [114, 115]. Nielsen [116] and Leumann [117, 118] further examined the importance of this carbonyl moiety and suggested that the amide geometry and/or its electrostatic interaction with adjacent residues contribute to the molecular recognition of PNAs for natural oligonucleotides.

Olefinic Polyamide Nucleic Acids (OPAs) Early work on molecular modelling [115, 123] suggested that intra- or inter-residue hydrogen bonds between the backbone and this tertiary amide group in the PNA strand might be important secondary structure-stabilising elements a statement that was not supported by the structural analyses (Fig. 22). NH

3'-OH

B

N

H

3'-OH O

N

O H N

NH

O 5'-P a Interresidue H bond

B

N

DNA

Therefore, it appears from the literature data that improvements in the specificity of DNA recognition by PNAs can be achieved by using chiral monomers with Dconfiguration, positive charge and a position in the middle of the PNA strand.

2575

In addition, NMR and X-ray crystallographic data have independently shown that in the bound conformation, the amide bond favours the rotamer, which orients the carbonyl oxygen towards the C-terminal of the peptide (formally the Z(O) geometry) in PNA/DNA [119, 120], PNA/RNA [121], as well as the PNA/PNA [122] complexes. However, the two rotamers of the amide are in rapid equilibrium in the free state of a single stranded PNA oligomer [119].

DNA

antiparallel/parallel preference in the complexation of DNA. Indeed, D-Lys-containing PNAs showed an increased preference for the antiparallel mode of complexation, whereas the L-Lys-PNAs slightly improved the stability of the parallel binding [111]. Improved mismatch recognition was also reported for D-Lys-containing PNAs [110]. Thus, it appears that the insertion of stereogenic centres derived from positively charged D-amino acids in suitable positions in the PNA strand can lead to more specific binding with complementary DNA, both in terms of complexation direction and of mismatch recognition. It has also been noted that the stereogenic centre was more efficient in affecting selectivity when it was positioned in the middle of the PNA strand [111].

Current Medicinal Chemistry, 2005, Vol. 12, No. 22

O

5'-P

b Intraresidue H bond

Fig. (22). Interresidue H-bond and intraresidue H-bond.

To eliminate ambiguity regarding the conformation and to elucidate the structural and electrostatic role of this amide group on DNA and RNA binding, Leumann et al. [124] have designed olefin polyamide nucleic acids (OPAs), in which this central amide functionality is replaced by an isostructural, configurationally stable C-C double bond in either the Z or E configuration (Fig. 23). The synthesis of chimeric PNA-oligomers containing OPA units was performed using the building blocks (E)/(Z)-76 and (E ) - 7 7 , and followed the MMTr/acyl (MMTr=monomethoxytrityl) protecting group strategy developed earlier for the synthesis of PNAs [125]. Two conclusions can be drawn from the Tm analysis (UV-melting curves) of the chimeric PNA/OPA oligomers paired to their antiparallel DNA complement. A geometric mismatch of the base-linker unit is associated with an energetic penalty on duplex formation. Stereochemical constriction of the base-linker unit into the pairing competent geometry of PNA does not increase but rather decreases affinity. Assuming that replacement of the amide functionality by the double bond has a minor influence on the conformational preferences of the rest of the PNA unit, there exists a considerable electrostatic contribution of the amide functionality to pairing. Further conclusions can be drawn with respect to molecular recognition of DNA by PNA. The amide functionality in the base-linker unit in PNAs determines significantly the affinity and preferred strand orientation in PNA/DNA duplexes. Furthermore, it seems to be responsible for the propensity to form PNA2-DNA triplexes.

2576 Current Medicinal Chemistry, 2005, Vol. 12, No. 22

O

Porcheddu and Giacomelli

HN O

HN

HN

Base Base

Base

N O O

P

Base

O

O

O

O

O

HN O

HN

HN

Base Base

Base N

O O

O

P

Base

O

O

O

O

O

HN

HN

HN PNA

DNA

O

(Z)-OPA

(E)-OPA

Fig (23). Sections of the chemical structures of deoxyribonucleic acids (DNAs), polyamide or peptide nucleic acids (PNAs), and (E)and (Z)- olefin polyamide nucleic acids ((E)- and (Z)-OPAs). T O

O

GPC

O

N H

MMTr

N H

MMTr

O

75

N H

O A(Bz)

T O MMTr

NHEt 3

(E)-76

N H

O O

NHEt3

M MTr

N H

(Z)-76

O

NHEt 3

(E)-77

Base: T = Thymine, A = Adenine

Fig. (24). (E)- and (Z)-OPA building blocks containing the base thymine.

With OPA, new and rather unexpected structure-activity relationships in PNA/DNA recognition could be obtained. Furthermore, OPA offers the unique possibility, with respect to PNA, to introduce a fourth substituent on the double bond. This could be of interest, for example, for improving binding or solubility properties, or for the attachment of functional units that may act on bound complementary RNA or DNA.

O HO

NH2

Base O

O

O N

Leumann reasoned that the differences in the recognition properties of OPA are mainly related not to conformational differences [116] relative to PNA, but rather to changes in the H-bonding capacity, electrostatic properties, or solvation. Consequently, the intriguing question arose as to what extent the dipole moment of the linker-carbonyl group in

Base

Base

HO

Fluorinated PNAs

O N

NH2

O

HO

NH2 (E)-OPA 78

PNA

Base

H

H

HO

NH2 (Z)-OPA 79

Base O

F

HO

NH2 (Z)-F-OPA 80

Fig. (25). Two rotamer forms of the PNA monomers and chemical structures of the (E)- and (Z)-OPA monomers and of the F-OPA monomer (bottom).

Peptide Nucleic Acids (PNAs)

Current Medicinal Chemistry, 2005, Vol. 12, No. 22

2577

PNA influences its binding properties. The OPA scaffold is ideally suited to address this question.

units relative to those of PNA. Dipole effects of the baselinker unit could be excluded.

Since a C-F bond is structurally and electrostatically a decent mimic of a C=O bond without hydrogen bonding capacity [126] and was often used in that respect [127], they decided to synthesise and investigate the pairing properties of the (Z)-F-OPA system (80). The (Z)-F-OPA system has the same geometric configuration as the (E)-OPA series (Fig. 25).

Numerous chemical modifications to the aegP N A backbone have demonstrated that small changes typically have detrimental effects on binding to complementary DNA and RNA sequences [129]. For example, the tertiary amides in the backbone are crucial for binding. Pioneering works of Nielsen’s group in this area demonstrated that changing one tertiary amide in the aegPNA backbone to a tertiary amine dramatically reduces the ability of the oligomers to bind DNA (93, Fig. 27) [32]. This work indicated that even the presence of favourable ionic interactions between the protonated amine and the oligonucleotide was not able to overcome the loss in structural rigidity. The secondary amides in an aegPNA backbone have been modified to a tertiary N-methyl amide, although moderate loss in binding to DNA is observed (94, Fig. 27) [130]. The work of Efimov et al. elegantly showed that the secondary amides of a e g PNA can be replaced with a phosphonate or phosphonamide, again with moderate reduction in binding to DNA (95, Fig. 27) [131].

Introduction of the fluorine atom at the vinyl position thus mimics the geometry and in part, the dipole moment of the carbonyl group in bound PNA. The Leumann’s group [128] has synthesised the novel fluorinated olefin peptide nucleic acid monomer (Z)-t-F-OPA (91) in 12 steps (Fig 26). The key steps being a lactonisation (lactone 85) as a stereo differentiating step in the elaboration of the double bond and a highly selective Mitsunobu substitution of an allyl over a homoallyl hydroxy function (diol 88) by the thymine base. Incorporation of (Z)-t-F-OPA (91) into PNA was achieved by the MMTr/acyl strategy. It was found that duplex stability strongly depends on the position of the modification within the strand. The reason for this is still unknown, but might likely be caused by the differential stacking interactions or solvation properties of the OPA O

OH

O

Recently, Appella et al. [132] are interested in examining the properties of modified PNAs where secondary amines in the backbone replace secondary amides. They refer to this type of oligomer as a polyamine nucleic acid (PANA). On the basis of Nielsen’s DNA binding studies with tertiary

OH

O b, c

a EtO

OEt

81

HO

OH

82

O O TrMMO

F

d TrMMO

OMMTr

83

O OEt

F

O

e

OMMTr

84

OH

F

g

RO f

h TBDMSO

T

OH

87

85 H 86 OTBDM S T

T

F

F i, j TBDMSO

OH

k, l HO

O

F

N3

88

HO

89

T = Thymine

T O

90

N3

m, n

F

HO

NMMTr 91

Rea gents and conditions: (a) LiAlH4, THF, rt, 4 h; (b) MMTrCl, DMAP, pyridine, rt, 8 h; (c) IBX, DMF, rt, 3 h; (d) n-Bu Li, (EtO)2P(O)CHFCO2Et, THF, -78 °C, 1.5 h, then 83, THF, -78 °C, rt, 5 h; (e) BCl 3, CH2Cl2, rt, 2 h. (f) TBDMSCl, imidazole, DMF, rt, 12 h (g) NaBH4, CeCl3. 7H2O, methan ol, 0 °C, rt, 4.5 h; (h) N3-benzoyl thymine, PPh3, DIAD, THF, rt, 12 h; (i) LiN3, CBr 4, PPh3, DMF, 0 °C, rt, 12 h; (j) TBAF, THF, rt, 5 h; (k ) Dess -Martin periodinane, CH2Cl 2, rt, 2 h; (l) NaClO2, NaH2PO4, 2-methyl-2- butene, tBuOH, rt, 12 h; (m) PPh3, pyridine, concentrated NH3, rt, 3 h; (n) MMTrCl, pyridine, rt, 12 h.

Fig. (26). Synthesis and incorporation into PNA of fluorinated olefin PNA (F-OPA) monomers.

2578 Current Medicinal Chemistry, 2005, Vol. 12, No. 22

92. aeg-PNA

Tertiary Amide

B O

B O

O

Good Binding to DNA

B

N

O O

95. Phosphonate and Phosphonamide PNA

N H

N

P

B O

B

N

PANA Portion

B

O

O

N H

N H

X = O, NH

B

N

N

X

Moderate Loss in DNA Binding

Moderate Loss in DNA Binding

O

O

O

Me

96. PNA-PANA

N H

B

O

O N

N

N H

B

O

O

N

Significant Loss in DNA Binding

B

O

O

N

N H

Secondary Amide

B O

93. Tertiary Amine PNA

N

N H

94. N-Methy l Amide PNA

B

O

O N

Porcheddu and Giacomelli

97. tcypPNA-PANA

N H

O

O N

O N

N H

N H

(S, S)-Cyclopentane diamine

PNA Portion

Fig. (27). aegPNA and associated backbone modifications.

amine PNA (93, Fig. 27) [133], increases in the flexibility of the PNA backbone are likely to decrease oligonucleotide binding. Therefore, they predicted that a PNA with one PANA unit (96, Fig. 27) would bind very poorly to DNA compared to the original, unmodified PNA. The synthesis of PANA oligomers requires a reductive amination on solid support between a primary amine of an oligomer on the resin (98a or 98b) and aldehyde (99a or 99b) (Fig 28). These conditions were used to make PNAPANA oligomers (100) and (101), each with a single PANA unit in the middle of a PNA oligomer. Surprisingly, UV melting curves of PNA-PANAs 100 and 101 show little deviation in oligonucleotide binding compared to the control PNAs. Even though PNA-PANA 100 was predicted to have very weak binding to C (Cbz)

Structural preorganisation of a synthetic oligomer into a conformation that closely resembles its DNA or RNA bound conformation is expected to increase the thermal stability of the complex (duplex or triplex) it would form with its complementary natural oligonucleotide. In recent years, a

O

O +

N

TCA

Aromatic Peptide Nucleic Acids

T O

O Lys

complementary oligonucleotides compared to the aegPNA, the melting curves looked almost identical [134]. The T m calculated from the first derivatives of the PNA-PANA:DNA melting curve is identical to the T m for the aegPNA:DNA duplex. Introduction of an (S, S)-transcyclopentane diamine into the PNA-PANA resulted in T m increases that are consistent with the benefits that this ring has on the corresponding aegPNA sequence.

NH2 98a

N

H

NHBoc

99a 15 eq.

1. Molecular Sieves 2. NABH(OAc) 3, 15 h 3. Dibenzyldicarbonate 4. Amide Couplings 5. Cleavage and Purificatio n

H2N-GTAGATCACT-Lys PNA-PANA 100

C (Cbz)

Lys

T

O

O

N (S, S) NH2 +

TCA 98b

O

O

(Same Conditions ) N

H

99b 15 eq.

Fig. (28). Introduction of PANA unit into a PNA oligomer.

NHBoc

H2N-GTAGATCtcypACT-Lys tcyp-PNA-PANA 101

Peptide Nucleic Acids (PNAs)

Current Medicinal Chemistry, 2005, Vol. 12, No. 22

number of rigidified PNA analogues have been described in the literature, aiming to address the conformational flexibility of the original PNA analogues. In 1997, Tsantrizos et al. reported the design and synthesis of the first peptide nucleic acid analogueue having an aromatic moiety as an integral part of its backbone, which they termed aromatic peptide nucleic acids (APNA) [135] (Fig 29). Preliminary hybridisation experiments with DNA and RNA identified monomer (102) as the most promising lead structure from this class of analogues [136, 137]. Unfortunately, the poor solubility observed with homopolymers of (102) prevented an in-depth evaluation of these analogues as potential antisense agents or as ligands for nucleic acid processing enzymes [137]. In a recently study, they have described the synthesis and some preliminary hybridisation properties of four new derivatives of 102, which were designed in order to (a) lock the aromatic backbone into a conformationally unique structure, while maintaining the same space distribution between the nucleobases and (b) modulate the electrostatic potential of the aromatic backbone of (102) in an attempt to improve the solubility of its homopolymers in aqueous media. RO

O

T

mN

O n

r NHR1 102 m = n = 1, r = 0 103 m = 1, n = 0, r = 1 104 m = 2, n = 0, r = 0

HO

O

T

N

O

2579

APNAs in aqueous media, and possibly alter their binding properties. PNA/APNA chimeras containing a monomer unit of (1 0 3 ), (1 0 5 ) or (1 0 6 ) were prepared and their hybridisation properties with DNA and RNA were evaluated. Based on the preliminary data, these structural alterations appear to be well tolerated, leading to only a minor decrease in the thermal stability of the triplexes formed with DNA or RNA. However, these minor effects may not accurately reflect the hybridisation properties of the corresponding APNA homopolymers. The more polar compounds (105) and (106) are of special interest, since their homopolymers are expected to be fairly soluble in aqueous media. These oligomers may be valuable tools in exploring possible inter-residue π–π interactions [141], such as those observed in protein folding [142], DNA duplex structures [143] or protein/nucleic acid interactions [144]. Plausible dipole/quadrupole interactions (i.e. π-cation or X–H– hydrogen bonds) [145] along the backbone of these oligomers may also contribute to the pre-organizing forces that favour helix formation, or binding to proteins, and will be explored. Ultimately, their ability to affect the biophysical properties of synthetic oligomers by modifying their structure and electrostatic potential is critical to our endeavour towards achieving a better understanding of the factors, which dictate molecular recognition between synthetic oligomers and natural oligonucleotides.

X NH2 R 102 R = H, X = CH 105 R = COOH, X = CH 106 R = H, X = N

Fig. (29). Aromatic peptide nucleic acids (APNAs).

Based on this literature precedence, Tsantrizos et al. [138] have focused their attention on replacing the 2aminobenzyl glycyl backbone of the APNA monomer (102) with a conformationally more rigid backbone such as the Nphenyl-N-alkyl amide derivatives (103) and (104). In most cases, N-phenyl-N-alkyl amides would be expected to adopt exclusively the E(O) geometry, with the plane of the phenyl moiety oriented perpendicularly to the plane of the amide bond [139]. Consequently, the N-alkyl unit of monomers (103) and (104) would be expected to extend in the Cterminal direction, having an amide conformation analogueous to the Z(O) rotamer preferred by PNAs for binding to natural oligonucleotides in either a duplex or triplex structure [140]. However, these new analogues were designed so as to maintain the space distribution between nucleobases in the same was as in the lead structure (102) (total of a 6-bond spacing between units, 5 s-bonds and 1 π -bond) in homopolymers of APNA. Furthermore, modifications to the electrostatic character of the 2-aminobenzyl ring of the lead structure (102) by the incorporation of an anionic carboxylate substituent or possibly a cationic pyridyl backbone (105 and 106, respectively), was expected to improve the solubility of

5. PNA WITH FIVE- OR SIX-MEMBERED CYCLIC STRUCTURES An effective way to preorganise the PNA strand for attaining hybridisation competent conformation is to conformationally rigidify the backbone by introducing bulky substituents or ring structures into the backbone [146-149]. Such approaches providing a conformational lock as in locked nucleic acids (LNAs) by Wengel et al. [150, 151] or freezing the conformations of six-membered rings as in

O

H B

O

O O

P

O LNA

a. Locked 3'-endo conformation B

B O

O

O O

O

O O

P

O O

Hexitol NA

P

OH O

Altritol NA b. Frozen 3'-endo conformation

Fig. (30). a) Locked 3'-endo conformation in LNA; b) Frozen 3'endo conformation in hexitol and altritol.

2580 Current Medicinal Chemistry, 2005, Vol. 12, No. 22

Porcheddu and Giacomelli

O

C3

NH α β

O B C2

δ

O O P

H (OH)

B

γ

χ2

N χ1

O

e

O 108

107 NH 2 β 1

B

HN 2 β

B

N

109 a

N

1 109 b

HN B N

2

1

O

O

O

O

O

Cis-(1R, 2S)

Cis-(1S, 2R)

Cis-(1S, 2R)

NH

β 2 O

1 110 a

B

β N O

110 b

O Cis-(1R, 2S)

Fig. (31). Structures of 107 (DNA or RNA), 108 aegPNA, 109 chPNA, 110 cpPNA.

hexitol nucleic acids (HNAs) [152] and carbocyclic nucleic acids such as cylohexanyl (CNA) [153] and cyclohexenyl (CeNA) [154] nucleic acids by Herdewijn et al. have resulted in elegant designs of preorganised, hybridisation-competent structures with remarkable success in oligonucleotide series (Fig. 30). The pioneering work of Eschenmoser et al. [155, 156] employing pyranosyl (p-RNA) and threofuranosyl (TNA) nucleic acids has shed light on the important conformational differences and the hybridisation properties of five- and sixmembered rings in a nucleic acid backbone. A simple strategy for rigidifying a PNA is to incorporate a cyclic ring [157] into the C2-C3 carbon-carbon bond of the PNA backbone (Fig. 3 1 ). The objectives of these modifications were to overcome the ambiguity in binding orientations of cDNA/RNA, to restrict conformational flexibility in backbone and impart features for selective RNA and DNA binding [148]. The approaches include introduction of chirality in the achiral PNA backbone to influence the orientation of cDNA binding and designing of cyclic analogueues to preorganise the PNA structure, an attribute that could entropically drive the duplex formation.

accompanied by a decrease in the entropy loss and a reduced gain in enthalpy.

HN

H 2N

O

HN

H 2N

HN

H 2N

O

111b

(1R,2R)-cyclohexane-1,2-d iamine

Fig. (32). (S,S)-cyclohexyl PNAs 111a and -(1R,2R)-cyclohexyl PNA 111b. Dihedral angle β

Table 3.

Base O

χ β

α N H

Bas e

Base

O

O N

The results of this study demonstrated that the PNAs derived from the trans-(S, S)-cyclohexyl PNAs (111a) hybridises with the complementary DNA with slightly weaker affinity compared to the unmodified PNA, while trans-(1R, 2R)-cyclohexyl PNA (111b) lacked such a property [159]. Moreover, thermodynamic data indicated that the DNA binding of the (1S, 2S) cyclohexyl PNA 111a is

111a

(1S,2S)-cyclohexane-1,2-diamine

1,2-Cyclohexyl PNAs In one of the earliest approaches, Nielsen and co-workers examined this strategy by incorporating a cyclohexane ring on the ethylenediamine segment of the backbone [158]. The facile synthesis of these PNAs started from the well-known and commercially available trans-1,2-diaminocyclohexane (available as a single enantiomer in either the (S,S) or (R,R) form) (Fig. 32).

HN

H 2N

ε γ N δ

O

O

N

N H

(CH2) n

O N H

PNA, n = 0 aeg-PNA; n = 3, cpPNA; n = 4, ch-PNA

Compound

α

β

γ

δ

χ

ε

PNA2-DNA

-103

73

70

93

1

-175

PNA-DNA

105

141

78

139

-3

151

PNA-RNA

170

67

79

84

4

-171

chPNA (1S, 2R)

128

-63

76

119

1.02

-175

chPNA(1R, 2S)

-129

66

-78

-119

-0.87

174

cpPNA(1S, 2R)

84

-24

86

90

0.89

165

cpPNA(1R, 2S)

-84

25

-86

-90

1.2

-165

aDihedral angles from monomer crystal structures: chPNA, cpPNA.

Peptide Nucleic Acids (PNAs)

Current Medicinal Chemistry, 2005, Vol. 12, No. 22

NH 2

NH α

β γ χ2 δ N 1 χ ε O O 108

NH 2 β 1 N

1 β

2581

B O O

B

N

B O

109, (1R, 2S/1S, 2R)

O 111, (1R, 2R/1S, 2S) NH NH

H

N H N

H

111 (trans-d iaxal)

H

109 (cis-axial, equatorial)

Fig. (33). PNA analogueues and chiral cyclohexyl PNA.

An analysis of the X-ray structural data of the PNA2 DNA triplex [160] and the PNA-DNA duplex [161] and NMR data of the PNA-RNA duplex [121] suggested that the dihedral angle β could be a key factor for the rational design of a preorganised PNA structure (Table 3). The preferred values for β in the PNA2-DNA triplex and the PNA-RNA duplex are in the range 65-70°, while that for the PNA-DNA duplex is about 140°, suggesting that it may be possible to impart DNA/RNA binding selectivity if β is restricted to 6570° through suitable chemical modifications.

RNA. In view of results on trans-(1S , 2S /1R , 2R)cyclohexyl PNAs (111a and 111b) and PNA-DNA/RNA duplex structural data, it occurred to us that if torsion angle β can be restricted to values in the range 60-70°, the resulting PNA may bind to RNA selectively over DNA. This is possible in a cis-1,2-disubstituted cyclohexyl system wherein the substituents are in axial-equatorial disposition (Fig. 33). In cyclohexyl PNAs, this corresponds to (1S, 2R/1R, 2S) configuration (109), wherein the torsion angle β would be around 65°. These may be better suited for incorporation into the PNA backbone to impart DNA/RNA discriminating properties (Table 3).

Molecular Dynamics simulations (using molecular mechanics calculations with the MM3 force field), performed on model structures of (1S,2S)- and (1R,2R)-cyclohexyl PNAs (111a) and (111b) showed the torsion angle β (NCH 2-CH 2-NHCO) to be close to 180°, corresponding to a trans-(1,2-diaxial) disposition (Fig. 33) [158]. This suggested that the trans biaxial geometry of the (1S,2S/1R,2R)-cyclohexyl PNA (111a or 111b) is far from the required PNA conformation and not suitable for facile hybridisation with either DNA or RNA targets.

With such a rationale, Ganesh et al. have recently reported [162, 163] cis-cyclohexyl PNA (chPNA) in which the axial-equatorial disposition of cis-1,2 substituents with β in the range 63-66° (opposite in sign for the two enantiomers 1S, 2R and 1R, 2S) showed a stereochemistrydependent preferential binding of the derived PNA-T oligomers to RNA as compared to DNA (Fig. 33). This was interesting in comparison to the earlier report on trans-1,2diaxial-substituted cyclohexyl system in which the dihedral angle β is 180° and hence unsuitable for forming hybrids with either DNA or RNA.

Classical PNA is very flexible and can attain different conformations to accommodate binding to both DNA and N3 O

i, ii O

112

N3

O

iii

N3

NHBoc

OH

N

O +

R

O

113 (1R/S, 2R/S), (R = C3H7)

R

1 13 (1S, 2S; major), (1R, 2R; minor)

OEt

114a

O O T 110a cis (1 S, 2 R)

iii

NHBoc N3

O

N3

O

OEt

iv R

113 (1S, 2S)

N OH O 114b (1S, 2S; major)

Reagents and Conditions : (i) NaN3, NH4Cl, aq ethanol, reflux, 18 h; (ii) n-butyric anhydride, dry pyridine, DMAP (cat.), rt, 16 h; (iii) Pseudomon as cepacia (lipase), phosphate buffer, pH 7.2, 2.5 h; (iv) NaOMe in M eOH.

Fig. (34). (1S, 2R/1R, 2S)-Aminocyclohexyl Glycyl Thymine PNA 110.

O

T 110b cis (1R, 2S)

2582 Current Medicinal Chemistry, 2005, Vol. 12, No. 22

Table 4.

Porcheddu and Giacomelli

UV-Melting Temperatures (Tm Values)a of PNA2-DNA Complexes PNA

DNA Ib

DNA IIb

Poly rA

Poly dA

115, H-TTTTTTTTTT-Lys-NH2

69.2

58.0 (-11.2)c

>80.0

72.4

116, H-TTTTTSRTTTTT-Lys-NH2

41.1

26.1 (-15.0)

77.2

61.5 (-15.7)

117, H-TTTTTSRTTTTT-Lys-NH2

45.0

29.1 (-16.1)

70.7

63.5 (-7.2)

118, H-TSRTTTTSRTTTTT-Lys-NH2

34.4

23.3 (-11.0)

64.4

53.7 (-10.7)

119, H-TRSTTTTRSTTTTT-Lys-NH2

37.5

25.7 (-12.0)

58.6

53.5 (-5.1)

a T = melting temperature (measured in the buffer 10 mM sodium phosphate, 100 mM NaCl, 0.1 mM EDTA, pH = 7.0), PNA -DNA complexes. b DNA I = m 2 5’d(CGCA 10 CGC); DNA II 5’d(CGCA 4 CA 5 CGC). Values in parentheses indicate the difference in T m as a result of the mismatch in DNA sequence. c Values in parentheses indicate difference in binding with RNA over DNA. The values reported here are the average of three independent experiments and are accurate to ±0.5 °C.

The synthesis of the optically pure monomers (110a and 110b) is achieved by stereoselective enzymatic hydrolysis of an intermediate ester (113) (Fig. 34). The chiral PNA oligomers were synthesised with (1S , 2R /1R , 2S ) aminocyclohexylglycyl thymine monomers in the centre and N-terminus of aegPNA. Hybridisation studies with complementary DNA and RNA sequences using UV-T m measurements indicate that PNA with (1S, 2R)-cyclohexyl stereochemistry enhances selective binding with RNA over DNA as compared to control aegPNA and PNA with the other (1R, 2S) isomer (Table 4).

adjustments to attain the necessary hybridisation competent conformations. Modelling studies of a corresponding cyclopentane indicated that this ring would be better suited to adopt the requisite C2-C3 dihedral angles in PNAs. Energy-minimised conformations of a trans-diequatorial cyclopentane ring possessed dihedral angles of 70-90°. In addition, the broad potential energy well suggested that the cyclopentane could adopt dihedral angles up to 160°. Based on these predictions, Appella et al. [165] embarked on a synthesis of (S, S)-trans-cyclopentane diamine (tcyp) (124) to determine if it was a suitable conformational restraint for a PNA.

However, a slightly lower binding affinity for triplex formation was seen with c h PNA in comparison to unmodified aegPNA [162]. They surmised that despite a favourable β , the substituted cyclohexyl ring is inherently too rigid as it gets locked up in either of the chair conformations.

They employed previous published synthesis to prepare and incorporate the cyclopentane into the PNA backbone [166, 167]. Apella’s procedure yields the same diamine, but with the two nitrogen atoms introduced in a stepwise fashion onto the five membered ring so that orthogonal protecting groups can be placed on the nitrogen atoms (Fig. 35).

1,2-Cyclopentyl PNAs

Using modified manual solid-phase peptide synthesis procedures, 21 [168] different PNAs were synthesised to probe the effects of cyclopentane incorporation into PNA. The effects of trans-cyclopentane incorporation on PNADNA stability were studied within a 10-residue, mixed-base PNA sequence that has been extensively studied in the

A relatively flexible system would be a cyclopentyl ring in which the characteristic interconvertible endo-e x o puckering that dictates the pseudoaxial/pseudoequatorial dispositions of substituents [164] may allow better torsional 1) AcOH, EtOH NH2 O

Me

Ph

Ph

Me NH

1) LiOH 2) H2, Pd/C

NHBoc

3) Boc 2O

120

NHBoc

3) Benzene, reflux COOH

2) NaCNBH3 COOEt

1) ClCOOEt, NEt3, THF, 0°C 2) NaN3, H2O, 0 to 25°C

121

COOEt

123

122

N

C

O

1) BnOH, CuCl

> 99% ee

2) DMF, 89% NHBoc COOH N B

O

126, B = T 127, B = C(Cbz) 128, B = A(Cbz)

1) B-CH2COOH, EDC-HCl, DMAP (cat), DM F 2) LiOH, THF/H2O

Fig. (35). (S, S)-trans-Cyclopentane-constrained peptide nucleic acids.

NHBoc COOMe HN 125

1) H2, Pd/C 2) BrCH2COOMe, NEt3, DMF

NHBoc 124 NHCbz

Peptide Nucleic Acids (PNAs)

Current Medicinal Chemistry, 2005, Vol. 12, No. 22

literature, by Nielsen and co-workers in particular (Table 5) [169, 170]. The tcyp-PNAs bind with higher affinity and better sequence specificity compared to unmodified PNA, and the incorporation of multiple cyclopentanes improves these properties further. From variable-concentration Tm data, the increase in DNA binding affinity of a cyclopentane-modified PNA is due largely to an entropic effect, indicating that replacing the ethylenediamine portion of a PNA with a carbocyclic ring significantly reduces the conformational flexibility of an unbound PNA, lowering the entropic cost associated with formation of a PNA-DNA complex [171, 172] (Table 5). Table 5.

Melting Temperature Data for PNA-DNA Complexes ∆Tm (°C)c

Entry

Sequence

Tm (°C) for DNAb

1

GTAGATCACT-Lys

48.9

2

GTAGAT∗CACT-Lys

54.9

6.0

3

GTAGATCA∗ CT-Lys

54.5

5.6

4

GTAGATC∗ ACT-Lys

54.2

5.3

5

GTAGAT∗ C∗ACT-Lys

60.2

11.3

6

GT∗ AGAT∗ CACT-Lys

59.6

10.7

7

GTAGA∗ T∗ C∗ ACT-Lys

63.2

14.3

8

GTAGA∗T∗ CA∗ CT-Lys

64.4

15.5

9

GT∗ AGA∗ T∗CA∗ CT-Lys

70.3

21.4

10

GTAGAT∗ RCACT-Lysd

na e

na e

11

GGCAGTGCCT-Lys

57.7

-

12

GGCAGT∗ GCCT-Lys

65.7

8.0

a Cyclopentane stereochemistry is (S, S), unless indicated otherwise. B* = tcyp residue. b Conditions for T m measurement: 150 mM NaCl, 10 Mm phosphate buffer, pH 7.0, 0.1 mM EDTA, UV measured at 260 nm from 90 to 25 °C, in 1 °C increments. All values are averages from two or more experiments. Approximate error for T m ’s is ±0.6 °C. c ∆T m is the difference in melting temperature between unmodified PNA (entry 1) and cyclopentane modified PNA, unless indicated otherwise. d B*R ) tcyp with (R, R) stereochemistry e No detectable melting transition observed.

The enthalpy of binding calculated from these data is consistent with a tcypPNA-DNA duplex that is similar in structure to an aegPNA-DNA duplex [173]. The stereochemistry and size of the ring incorporated at this position also impact the ability of PNA to bind DNA. For instance, previously published studies that examined the effects of replacing the ethylenediamine portions of PNAs with trans-cyclohexane did not report the same benefits as Appella reports for trans-cyclopentane [173]. Furthermore, both studies have found that the (S, S) stereochemistry in five- and six-membered rings is more compatible than (R, R) in the PNA-DNA duplex. They speculate that, in order for a carbocyclic ring to promote PNA binding to oligonucleotides, both ring size and stereochemistry must restrict the PNA to access only the range of dihedral angles necessary for binding, while excluding conformations that are irrelevant to duplex

2583

formation. The success of trans-cyclopentane compared to trans-cyclohexane is most likely due to conformational differences between the two rings. At the same time, Ganesh et al. [174] have presented interesting results on the stereospecific synthesis of cis-(1S, 2R/1R, 2S)-cpPNA thymine monomers, position wise incorporation into homothymine aegPNA oligomers, and DNA/RNA hybridisation studies. The synthesis of the enantiomerically pure 1,2-cis-cyclopentyl PNA monomers (148a and 148b) was achieved by stereoselective enzymatic hydrolysis of a key intermediate ester (140) (Fig. 36) The chiral (1S, 2R/1R, 2S)-aminocyclopentylglycyl thymine monomers were incorporated into PNA oligomers at defined positions and through the entire sequence. The torsion angles β as deduced from the X-ray crystal structures [175] for [(1S, 2R)-148a] and [(1R, 2S)-148b] are -24 °C and +25°C, respectively, which are much less than that found in PNA2-DNA and PNA-RNA complexes and cis-cyclohexyl systems (Fig. 36). In solution, the tertiary amide bond in PNA is known to exist as a rotameric mixture [176]. In the present structures, the amide bond is trans with carbonyl pointing towards the C terminus. The crystal structure data also shows pseudoaxial-psuedoequatorial dispositions for the cis-1,2substituents in (1S , 2R/1R , 2S)-cyclopentyl PNA monomers, with the bulky substituent carrying the nucleobase directed into a pseudoequatorial position. These monomers were built into PNA oligomers by solid-phase peptide synthesis; UV melting temperatures of the cpPNAs with complementary and mismatched DNA and RNA indicated that cpPNAs having a (1S, 2R)-cyclopentyl unit have a higher binding affinity to RNA than DNA, and PNA oligomers with (1R, 2S)-cyclopentyl units showed relatively higher affinity towards DNA. The stereo preferences in c p PNA binding of DNA/RNA are accompanied by overall enhanced binding affinities compared to a e g - PNA, unlike c h PNA, where stereochemistry-dependent discrimination of DNA/RNA was observed but with lower Tm values. Cis-cpPNAs show high mismatch discrimination compared to aegPNAs, revealing base-specific recognition. The torsional angle β in a 1,2-disubstituted c i s cyclopentyl system is less than that in chPNA, but seems to have significant consequences on the hybridisation ability of cpPNA oligomers. The higher binding of cyclopentyl PNAs is perhaps a consequence of the relative ease of conformational adjustments in a cyclopentyl ring as compared to rigid locking of cyclohexane systems. The favourable conformational features of the monomers are cooperatively transmitted to the oligomeric level. The results of cis-cpPNAs presented here support the idea of improving the stability and DNA/RNA binding selectivity via rational conformational tuning of classical PNA to achieve preorganisation. The enhanced thermal stabilities observed for aeg-cpPNA and cpPNA complexes with DNA are not at the cost of losing base pairing specificities. This is borne by data for their complexation with DNA IV [dA 4CA 3] that carries a single mismatch at the middle position (Table 6). The lower mismatch tolerance and higher binding affinities to matched

2584 Current Medicinal Chemistry, 2005, Vol. 12, No. 22

Porcheddu and Giacomelli

NBoc

N

NBoc xiii

COOH

N

B

B

O

COOEt 148b

O

149b v-xii

2 N3

N3 O

i, ii

O R 140 (1R/S, 2R/S) R = C 3H7)

141a (1R, 2R)

iii

O

+ 1 OH

O

129

N3

N3

iii

O

N3 iv

O O

R

140 (1S, 2S major) (1R, 2R, minor)

OH

R

141b (1S, 2S)

1 40 (1S, 2S)

v, vi NHBoc vii OH

142

NHBoc

NHBoc viii 143

NHBoc ix

OMs

N3

144

x

NHBoc

NHBoc NHBoc

N B

NH2

145

xiii

COOH

N B

O

xii

COOEt O

NHBoc xi

148a

Cl

149a

147

N

COOEt

146

N H

COOEt

O

Rea gents and conditions: (i) NaN3, NH4Cl, ethanol/water (2:8) reflux, 16 h . (ii) n-Butyric anhydride, dry pyridine, DMAP (cat.), rt, 16 h. (iii) P . cepacia (lipase), phosphate buffer, pH 7.2, 1.5 h. (iv) NaOMe in MeOH. (v) PtO2, dry EtOAc, H2, 35-40 psi, rt, 3.5 h. (vi) (Boc) 2O. (vii) MsCl, dry triethylamine, rt, 0. 5 h. (viii) NaN3, dry DMF, 70 °C, 5 h. (ix) PtO2, MeOH, H2, 35-40 psi, rt, 3.5 h. (x) BrCH2COOEt, KF-Celite, dry CH3CN, 65 °C, 4h. (xi) ClCH2COCl, Na 2CO3, Dioxan/H2O (1:1), 0 °C, 5 min. (xii) Thymine, K2CO3 dry DMF, 65 °C, 4 h. (xiii) 0.5 M LiOH, aq THF, 0.5 h

Fig. (36). Synthesis of (1S, 2R/1R, 2S)-cis-Cyclopentyl PNAs (cpPNAs) chimera.

complementary DNA III sequence reflects the sequence specific recognition of cpPNAs. The recent success with cis-cyclopentane in stabilising oligothymine PNA2 -DNA triplexes indicates that the cis Table 6.

stereochemistry about the cyclopentane ring is also compatible with the requisite conformations to promote PNA binding to DNA. However, further work will be required to determine whether the cis- or trans-cyclopentane

UV-Tm of cpPNA:DNA/RNA Triplexesa PNA

DNA III

∆ T m )b DNA IV (∆

Poly rA

aegPNA 150, H-TTTTTTTT-LysNH2

48.9

34.5(10.5)

62.0

cpPNA 151, H-TTTTTTTtSR-LysNH2

54.9

27.8(23.2)

73.5

cpPNA 152, H-TTTtSRTTTT-LysNH2

54.5

11.0(11.0)

76.0

cpPNA 152, H-tSRTTTTTTT-LysNH2

54.2

26.4(18.1)

66.0

cpPNA 153, H-TTTTTTTtRS-LysNH2

60.2

28.0(27.0)

>85.0

cpPNA 154, H-TTTtRSTTTT-LysNH2

59.6

32.0(30.0)

61.0

cpPNA 155, H-tRSTTTTTTT-LysNH2

63.2

27.8(20.9)

69.0

cpPNA 156, H-(tSR)8-LysNH2

64.4

Nd

>85.0

cpPNA 157, H-(tRS)8-LysNH2

70.3

Nd

>85.0

a All values are an average of three independent experiments and accurate to within ±0.5°. DNA III, d(CGCAAAAAAAACGC); DNA IV = d(CGCAAAACAAACGC). Buffer: sodium phosphate (10 mM), pH 7.0 with 100 mM NaCl and 0.1 mM EDTA; nd, transition not detected. bMismatch destabilization.

Peptide Nucleic Acids (PNAs)

Current Medicinal Chemistry, 2005, Vol. 12, No. 22 TOTU

COOH N 158

N

+

DIEA, DMF, 4h O

H N

BocHN

2585

N

O

N

O

N BocHN

OMe 160

OMe

159 H2O, 4h

N

NaOH, EtOH

O

N BocHN

N 161

O OH

Fig. (37). PNA as a scaffold for metal ions.

stereochemistry is the most beneficial in terms of stability and sequence specificity for a large number of different PNADNA duplexes. 6. PEPTIDE NUCLEIC COMPLEX CONJUGATES

ACID

AND

METAL

Recently, ligands have been introduced in DNA strands, and metal ions have been used to bridge these modified DNA strands into duplexes or triplexes. Substitution of a natural base pair by a pair of ligands leads to DNA duplex destabilisation comparable to the destabilisation induced by the presence of a mismatch. Incorporation of metal ions into the ligand-modified DNA duplexes leads to formation of coordinative bonds that act in a manner analogueous to that of hydrogen bonds in DNA [177]. Recently, Achim et al. [177] have demonstrated that metal incorporation can be expanded to peptide nucleic acid (PNA). Some of the advantages of using PNA as a scaffold for metal ions are the greater chemical stability of amide bonds of the PNA backbone as compared to the phosphate and glycoside bonds of DNA, and the fact that the neutral PNA backbone offers more flexibility for controlling the overall charge of metal-containing structures than does the anionic DNA backbone. Also, to create PNA monomers, ligands can be attached to the secondary amino group of aeg by acylation, a synthetic method simpler than the ones for making ligand-containing phosphoramidites for DNA synthesis. Bipyridine Ligands Achim has selected bipyridine (158) as ligand, which has a high affinity for metal ions, in order to avoid the possibility that the amide groups in the backbone and the nucleobases might act as competing chelating sites for metal ions (Fig. 37). The monomer was incorporated in PNA oligomers using solid phase peptide synthesis and Boc-protection strategy. They have replaced two nucleobases situated in

complementary positions in the middle of a 10-bp PNA duplex (162, M = A, N = T) with bipyridine (163, M = N = bipy) (Fig. 38). They have investigated the interaction of 162 and 163 with Ni2+ , Pd2+ , and Pt2+ because they can form square-planar complexes. Substitution of bipyridine for a nucleobase leads to modified peptide nucleic acid (PNA) single strands that are bridged in the presence of Ni2+ into a duplex containing a combination of hydrogen and coordinative bonds. H-GTAGMTCACT-LysNH2 (162) NH2-Lys-CATCNAGTGA-H (163)

Fig. (38). PNA sequences.

CD experiments demonstrate that the duplex adopts a structure similar to that of an unmodified 10-bp PNA duplex, and UV melting experiments show a very sensitive dependence of the duplex stability on the substitution of a nucleobase pair with a pair of ligands or a metal-ligand alternative base pair. Modified Naphthalene Diimide with PNA-DNA Duplex In future therapeutical applications, it might be of advantage if PNA can be activated only in specific cells, thus avoiding the risk of adverse side effects in non-targeted cells. One of the approaches for this involves targeted delivery of PNA to specific cells by tethering PNA to the ligands recognised by cell receptors [178, 179]. Another is the activation of PNA by the component present in the cell. Mokhir et al. have shown for that Zn2+ [180] can substantially increase binding affinity of terminally modified PNA to DNA targets in vitro. Zn2+ is present at high concentrations in brain [181], pancreas [182], and spermatozoa [183]. In breast cancer, tissues zinc levels are increased by 72% [184]. They have synthesised PNA in which a Zn2+ chelating ligand is conjugated via an aromatic linker. Intercalation of the aromatic linker is regulated by the metal coordination to the ligand (Fig. 39). It has been recently shown that unsymmetrically modified naphthalene diimide (NADI) intercalates within

2586 Current Medicinal Chemistry, 2005, Vol. 12, No. 22

Porcheddu and Giacomelli

O NH N

L

O

O

O

N N

O NH

O

N

NADI

N

N

T

O

O

L=

NH NH

O

NH

HN

N

HN

O O

N

N L1

N

NH2

C

O

L2

N O

O PNA

16 3. 16 3a. 16 4. 16 5. 16 6. 16 6a.

DNA (3'-5')

L1-NADI-gly-TCACAACTA-Lys3 L1-NADI-gly-CATTCG-Lys3 L1-gly-TCACAACTA-Lys 3 L2-NADI-gly-TCACAACTA-Lys3 NADI-gly-TCACAACTA-Lys3 TCACAACTA-Lys3

167. ACCAGTGTTGATTCT 168. ACCGTAAGCTCT 170. ACAGTAAGCTCT 171. ACGGTAAGCTCT 172. GTAAGC

Fig. (39). N-Terminally modified PNA. NADI (naphthalene diimide residue).

PNA-DNA duplex and strongly stabilises it [185, 186]. Therefore, this aromatic fragment was chosen as a linker between PNA and the ligand (Fig. 39). As ligands they have selected strong Zn2+ binders: bis(picolyl)amine (L1) and 1,4,8,11-tetraazacyclotetradecane (L2). Micromolar Zn2+ increases the affinity of the PNA probes to single-stranded DNA significantly, which is reflected in the best case in a 12.2 °C increase of T m upon addition of Zn2+ (Fig. 40). These probes hybridise to DNA within a few minutes at 22 °C. Cooperative binding of naphthalene diimide and the Zn2+ complex is more stabilising than binding of the Zn2+ complex alone. Stabilisation of PNA-DNA duplex by the terminal Zn2+-L1-NADI residue is more pronounced for a shorter PNA. The Zn2+-induced Tm increase for a duplex of (163) with the DNA, which has a mutation G-T in the penultimate position is reduced by 5.3 °C in comparison with that for a duplex of (163) with complementary DNA. No increase of PNA probe

B

A + Zn2+ Target DNA Zn2+

Fig. (40). Zn2+ -dependent binding of PNA probes to target DNA. Intercalator is shown as a thick line.

T m is observed for duplexes of (163) with DNAs, which have other internal mutations. These data indicate that NADI-L1-Zn 2+ is positioned between the terminal and penultimate base pairs (Fig. 40B). Since intracellular concentration of unbound transition metals is dependent on cell type [187], metal-responsive PNA–DNA/RNA binding can be employed for selective suppression of gene expression in these specific cells. Kraemer et al. [188] have chosen to covalently attach to the N-terminus of PNA bi- and tri-dentate ligands (Fig. 41) capable of forming 1:1 complexes with Zn(II), Cu(II) and NiII. 1:1 Metal complexes with these ligands have free coordination sites, which can enable direct coordinative interaction of metal ion with, for example, N-atoms of DNA nucleosides or O-atoms of phosphodiester groups of the DNA backbone. The ligands were attached to PNA via linkers of different lengths for optimisation of metal complex–DNA interaction (Fig. 41). Conjugates of peptide nucleic acids (PNA) and metal binding ligands were prepared using solid-phase synthesis. Stability of duplexes of bis-picolylamine–PNA conjugates and DNA was found to be modulated by equimolar concentrations of bioavailable metal ions: Ni2+, Zn2+>Cu2+. The better stabilisation of the duplex by Ni(II) and Zn(II) could indeed be a consequence of less hindered coordinative interaction of these metal ions with phosphodiester groups of the backbone of the target DNA (Fig. 42).

Peptide Nucleic Acids (PNAs)

Current Medicinal Chemistry, 2005, Vol. 12, No. 22

HN

O H N

O N

H N

O

N

T C (Bhoc)

NH O

O

T C(Bhoc)

R n O

O

N

N O

O

O

O

T C(Bhoc)

A(Bhoc)

A

C(Bhoc) A(Bhoc)

a

173

C

A(Bhoc)

A(Bhoc) A(Bhoc)

C(Bhoc) T

C(Bhoc) T

A(Bhoc) (LysBhoc) 3

A(Bhoc) (LysBhoc ) 3 177. n = 178. n = 179. n = 180. n =

176 d

1; R 2; R 1; R 2; R

=L 1 =L 1 =L 2 =L 2

N L2 NH N

f O H N

(Bhoc)

A (LysBhoc) 3

T C (Bhoc)

n O

175

184. TCACAACTA-Lys3 (PNA N-C) 185. TCTTAGTTGTGACCA (DNA 5'-3') 186. TCTTAGTTGTGAACA (DNA 5'-3') 187. TCTTAGTTGTGCCCA (DNA 5'-3') 188. TCTTAGTTGTTACCA (DNA 5'-3')

C (Bhoc) A(Bhoc)

O

Br

A(Bhoc) 174

N

H

(Bhoc)

(Bhoc)

n time b,c

n

N

C(Bhoc) T

A(Bhoc) (LysBhoc )3

H N

T C(Bhoc)

L1

A(Bhoc) A(Bhoc)

e

A(Bhoc) C (Bhoc) T

NH2

N

C(Bhoc)

C (Bhoc) A(Bhoc) N

R n O

A(Bhoc)

A(Bhoc)

HN

NH2

2587

g,h, e

A(Bhoc) C (Bhoc) T A(Bhoc) (LysBhoc )3

H N T C(Bhoc) A

N n O

L3

N N

(Bhoc)

C(Bhoc) A(Bhoc) A(Bhoc) C(Bhoc) T

182. n = 1 183. n = 2

A(Bhoc) (LysBhoc ) 3

181 Reagents and Conditions: (a) PNA s ynthesis; (b) Fmoc-Gly-OH, HBTU, HOBT, DIEA, DMF; (c) piperidine, DMF; (d) RCO2H, HBTU, HOBT, DIEA, DMF; (e) TFA, m-cresol; (g) bromoacetylbromide, DIEA, DMF; (h) di-(2-picolyl)amine, DIEA, DMF.

Fig. (41). Synthesis of PNA, which are N-terminally modified with metal binding ligands.

Sequence specificity of PNA was not compromised in the presence of these metal ions. N

H N

N O

HN

Zn2+ N

O

O

O

O

P O

T

A 189

PNA

DNA

Fig. (42). Proposed approach for metal-dependent binding of PNA probes to oligonucleotide targets.

Restriction endonucleases are enzymes that hydrolyse the phosphodiester backbone of deoxynucleic acids (DNA) sequence specifically. High selectivity and efficiency are characteristics of these enzymes, but they recognise only short oligonucleotide sequences, producing many fragments upon cleavage of genomic DNA. Therefore, it is desirable to develop artificial nucleases with higher sequence specificity. Artificial nucleases cleave nucleic acids by either an oxidative or hydrolytic mechanism. The former is observed with redox-active metal complexes [189], the latter one is based on the Lewis-acidity of metal ions [190]. An important drawback of the oxidative cleavage is the generation of products with unnatural ends, whereas the enzymes can further process the cleaving products of hydrolytical nuclease [189, 190]. Efficient hydrolytic

2588 Current Medicinal Chemistry, 2005, Vol. 12, No. 22

Porcheddu and Giacomelli

cleavage of linear DNA is only possible with strong Lewis acids such as Ce(IV) [191], Zr-(IV) [192], Th(IV) [193], and Co(III) [194].

acid (195 = L5-H, Fig. 44), protected iminodiacetate derivatives using HBTU activator (Fig. 44), or by coupling 4-chloromethylbenzoic acid to the N-terminus of the PNA followed by amination of the resulting PNA.

Conjugates of peptide nucleic acids and Zr-(IV) complexes hydrolyse complementary single-stranded DNA in a sequence selective fashion (Fig. 43).

Activity is strongly dependent on the nature of chelating ligand. There is significant no selective background cleavage by excess free Zr(IV) in single stranded DNA regions while DNA is protected within the PNA/DNA duplex. For the optimisation of Zr(IV)-based artificial restriction enzymes, chelating ligands that form stronger complexes with Zr(IV) without loss of phosphodiesterase activity are required.

O HN

O

Zr O

5'TAGTTGTGA C

O

P

190

O

OH

O CATCT3'

O

7. TERMINAL PNA CONJUGATES

Fig. (43). A proposed model for hydrolytic DNA scission by PNA-Zr-(IV) conjugate.

Homopyrimidine peptide nucleic acid oligomers (PNAs) bind sequence complementary targets in duplex DNA with high affinity and sequence specificity [149] by helix invasion forming triplex P-loop complexes. However, the

A series of ligands (Fig. 44) was attached by Kramer et al. [195] to the N-terminus of PNA either by coupling of the commercially available 8-hydroxy-quinoline-2-carboxylic O HO

O

HO

O

HO

O

O

O

OH O

OH

OH

191. n = 1, L1 192. n = 2, L2

n

N

N

N

194. L4

193. L 3 O

O

195. L5 H N

OH

OH

NH

HO

CH2

O H

OH

HO

N OH

196 a.L7

H

O

H

HO

OH OH NH2 OH

OH H

HO

OH

OH

OH

199. L10

NH2 198. L9

OH CH2OH

196. L6

197. L8

O HN N

O

N

NH O

O NH2

O NH2

PNA(PG) a

R1 b

NH PNA

O

C

HN PNA(PG )

O d

Cl

200. R 201. R 202. R 203. R 204. R 205. R

= L 1; = L 2; = L 3; = L 4; = L 5; = L6

206. R = 207. R = 208. R = 209. R = 210. R =

L7 L8 L9 L10 H

HN PNA(PG)

R2

Rea gents and conditions: (a) PNA synthesis; (b) (1) R1-OH, HBTU, HOBT, DIEA, DMF; (2) TFA/m-cresol; (c) 4-chloromethyl-ben zoic acid , HBTU, HOBT, DIEA, DM F; (d) (1) NaI; DMSO, H-R 2; (2) TFA/m-cresol. (PG = Bhoc for nucleobases, Boc for lysine.

Fig. (44). Modifiers of termini of PNA.

Peptide Nucleic Acids (PNAs)

Current Medicinal Chemistry, 2005, Vol. 12, No. 22

a) H N HN

PNA O

N

2589

b) PNA 211 (+4) Acr-Lys-TTTTTTTTTT-(egl)3-TTTLys TTTTLysTTT-NH2 PNA 212 (+4) H-Lys-TTTTTTTTTT-(egl) 3-TTTLysTTTTLysTTT-NH2 PNA 213 (+3) H-TTTTTTTTTT-(egl)3-TTTLysTTTTLys TTT-NH2 PNA 214 (+3) Acr-TTTLys TTTTLysTTT-NH2 PNA 215 (+4) H-Lys-TTTLysTTTTLys TTT-NH2

Fig. (45). (a) Chemical structure of the acridine moiety. (b) Structure and charge at pH 6.5 of the PNAs. Abbreviations: Acr (9aminoacridine), egl (8-amino-2,6-dioxaoctanoic acid), Lys (lysine), T (thymine), TLys (thymine monomer having D-lysine instead of glycine in the backbone).

helix invasion mechanism makes the binding very sensitive to elevation of ionic strength as this stabilises the target DNA duplex, and PNAs cannot effectively bind their target at physiologically relevant ionic conditions [196, 197]. Therefore, novel PNA constructs with improved helix invasion properties at elevated ionic strength are warranted. It has been shown that positively charged PNAs bind their target in duplex DNA with significantly increased efficiency [198]. Acridine Conjugated PNAs Nielsen and Bentin [199] reasoned that using a DNA intercalator could attain a similar and expectedly less ionic strength sensitive effect. To address this, the DNA intercalator 9-aminoacridine was conjugated to helix invading PNAs, and the duplex DNA binding efficiency of such constructs was measured at different ionic strength conditions by electrophoretic mobility shift analysis (Fig. 45). Remarkably, at physiologically relevant ionic strength (140 mM K + /10 mM Na + , 2 mM Mg2 + ), acridine conjugated PNAs showed 20-150-fold superior binding to a cognate sequence target as compared to the conventional PNAs. This enhancer effect of the 9-aminoacridine was even more pronounced in the case of one-stranded mono-PNAs, under which PNA (without intercalator) showed hardly any binding. The enhancement occurred without compromising the sequence specificity of binding. Thus, simply conjugating the DNA intercalator 9-aminoacridine to PNA represents a major step towards the development of helix invading constructs for in vivo applications such as gene targeting. Ester-Linked Pyrene and Adamantyl PNA Conjugates Cellular delivery of PNA is an area of active research [200-203], and several methods have been devised relying on conjugation to peptide transporters [204, 205] or accomplished via cationic liposome carriers [206-208]. In the latter case, either PNA-DNA hybrids or PNA-fatty acid conjugates were employed. Nielsen et al. have reasoned that it would be advantageous to develop chemistry that would enable incorporation of an enzyme cleavable ester function between the PNA and the carrier. For instance, this should

allow cellular (and animal) delivery of lipophilic PNA prodrugs that could transverse the lipid bilayer of the cell with subsequent intracellular enzymatic release of the free PNA. However, PNA oligomerisation (and especially cleavage from the resin) involves rather harsh acid and/or alkaline treatment, which is not a priori compatible with ester stability. Nonetheless, Nielsen et al. [209] have described the synthesis of a Boc-protected amino acid containing an ester function, 2-([N-Boc-glycyl]oxymethyl)benzoic acid, which has been incorporated into peptide nucleic acid (PNA) oligomers using standard solid-phase polymerization (Fig. 46)

O O

N H 216

PNA O

O O

N H O

PNA 217

O

Fig. (46). Structures of ester-linked pyrene and adamantyl PNA conjugates.

In model experiments, it is found that the ester is fairly stable in aqueous solution at pH 7.4 and 37 °C (t1/2 = 6 h), whereas it is rapidly cleaved in mouse serum and in kidney and liver homogenates (t1/2 = 0.1-0.5 min). Furthermore, ester-linked fatty acid PNA conjugates targeted to an aberrant splice site in luciferase mRNA were prepared and shown to be twice as potent for inducing active luciferase as the corresponding conjugate not containing the linker. Synthesis of PNAs Conjugated To TBTP by a Disulphide Bond Peptide nucleic acids are effective antisense reagents that bind specific mRNAs preventing their translation. However, PNAs cannot cross cell membranes, hampering delivery to

2590 Current Medicinal Chemistry, 2005, Vol. 12, No. 22

PNA

Porcheddu and Giacomelli

SH + P

S

S

PNA

P

bisTBTP

S

P

PNAssTBTP 219

218

PNA

S

S

S

P 219

Estracellular enviromental

+ -

ψp Dψ

cytoplasm 2GSH glutathion reductase

SH

P 219a

Slow efflux

GSSG

PNA

SH mithocondrion

PNA

SH AAAAAAA mRNA

Gene Expression A : The synthesis of PNAssTBTP by thiol-disulphide exchangeon incubating excess PNA with bisTBTP is shown. B : The lipophilic TBTP cation enables rapid transport of the PNAssTBTP conju gate across the plasma membrane.In the cytoplasm the disulphide bond is reduced by the endogenous glutathione pool, leaving the PNA free in the cytoplasm.The TBTP cation will initially distribute to the mitochondria and from there slo wly wash out of the cell.

Fig. (47).Synthesis and uptake of PNAs conjugated to TBTP by a disulphide bond. cells. To overcome this problem, Murphy et al. [210] have made PNAs membrane-permeant by conjugation to the lipophilic triphenylphosphonium (TPP) cation through a disulphide bond (Fig. 48). This procedure can be carried out by the conventional automated synthesis of a PNA containing a free thiol. The crude synthetic product is then incubated with excess bis-TBTP followed by a single reverse-phase HPLC purification to give the final PNAs TBTP. Disulphide conjugation of TBTP to a PNA led to efficient PNA uptake into the cytosol, where the disulphide bond was reduced and the bioactive antisense PNA was retained in the cell. This procedure is far more

straightforward than covalently linking a cell-permeant peptide to a PNA, or the alternative delivery strategy of first annealing the PNA to a complementary DNA to enable it to bind to cationic detergents [211]. Delivery facilitated by cationic detergents is also strongly cell type- and PNA sequence-dependent. This approach will greatly simplify the in vitro applications of PNAs as antisense reagents. Furthermore, as the disulphide is relatively stable in the extra cellular environment, it should be possible to use this approach to deliver PNAs to cells in vivo, facilitating pharmacological applications of PNAs.

Peptide Nucleic Acids (PNAs)

Current Medicinal Chemistry, 2005, Vol. 12, No. 22

N H

Lac

SPACE

B

O H N

N H O

N H

Antisense Moiety

Lac

O H N

n

2591

O N H

m

Labelling Moiety O

O

R2

N

N R1

13

PNA (CH2) 4

O

H N

HN N H

Harming Moiety

O

Lac

HN

Lac

NH2

N H

La c

n (CH2) 4

O

OH OH O

NH Lac PNA 220: R1 = H, m = 2; n = 2; R2 = H

Lac =

O

HO O

O H

S

N H

S HS

m = 2; n = 2; R2 = H

O N

R2 =

PNA 222: R1 = H, m = 2; n = 2; R2 = H

OH

OH

H 2N

NH2

PNA 221: R1 =

OH O

Rodamine

O

Fig. (48). Structures of lactose-PNA conjugates 220, 221 and 222.

conjugate targeted to the RNA template of telomerase inhibits telomerase, but only at high concentrations. These results suggest that lactose–PNA conjugates can specifically enter liver cells, but that they are compartmentalised and their efficient release into the cytoplasm is hindered. It is important to note, however, that oligonucleotides have been noted to possess much more favourable cellular uptake properties in animals relative to cell culture.

Lactose-PNA Conjugates Recently, Corey et al. [212] have developed two simple strategies for the attachment of lactose to PNAs (Fig. 49). Lactose can be recognised by the hepatic asialoglycoprotein receptor (ASGP-R) [213]. In their first approach, they added several lysines to the N-terminus of PNA. Three additional lysines were then added to both the α- and ε-amines of the first lysine. A total of eight lactose moieties were then coupled by reductive amination to afford PNA (220); two on the α-amines, and the remaining six on the ε-amines (Fig. 49). As a second strategy, they synthesised the branched lysine rich peptide separately and included a C-terminal cysteine. They then labelled this peptide with lactose by reductive amination and coupled it to cysteine-containing PNA by disulfide exchange to afford PNA (221).

8. PNAs CONTAINING NON-STANDARD NUCLEOBASES Alternative Nucleobases Increase in affinity between nucleic acids strands is mainly achieved by improving stacking interactions and hydrogen bonding [214]. Enhanced stacking can be accomplished by introducing polycyclic base analogueues,

Lactose–PNA conjugates enter cells that express ASGPR, but not cells that lack the receptor. A lactose–PNA H a)

H H

H

N

H N

N H N dR

O

dR

N

N H

H

H

N

N

O

N

N

N

H

N

H dR

N

N

dR

H N

N

O

dR

N N H

O

N

H N

223

H

O

N

N

H H

S

O H

O

N O

N O

N

H

b)

N O

N

N H

O 224

225 Boc

N H

N

COOH

Fig. (49). Hydrogen bonding in base-pairs of guanine with cytosine analogueues: (a) amino-G-clamp, (b) guanidino-G-clamp, and (c) the fluorescent phenothiazine PNA monomer.

2592 Current Medicinal Chemistry, 2005, Vol. 12, No. 22

Porcheddu and Giacomelli N

N

Cbz N

H

O

O

H

N

O Br

O

N H

O O

Fmoc

N H

N

N O

NH

N N

Cbz Cbz

N H

H H

N

O

N

O O

226 COOH

Fmo c

225

N

N H

COOH

227

Fig. (50). Protected amino-G-clamp and guanidine-G-clamp PNA monomers.

and an increase in the number of hydrogen bonds achieved by the simultaneous recognition of both the Watson-Crick and Hoogsteen binding faces of guanine and adenine bases.

Actually, there is increasing interest in modulating and expanding the recognition motifs [220] of standard base pairs, as this may have potential applications in diagnostics and nanomaterial chemistry [221, 222]. Employing no natural nucleobase ligands in place of natural nucleobases would help in understanding the recognition process in terms of various factors contributing to the complementation events such as hydrogen bonding and internucleobase stacking [223].

For this purpose, a tricyclic cytosine analogue, having the structure of an aminoethoxy-derivatized phenoxazine ring, was designed by Pedroso et al. [215] to bind to the guanine residue like a clamp (amino-G-clamp, Fig. 49a), and incorporated into oligodeoxynucleotides [216]. The protected amino-G-clamp and guanidine-G-clamp PNA monomers (225) and (227) have been obtained in eight steps from 5-bromouracil (226).

The non-standard nucleobases employed so far with PNA include 2-aminopurine [224] , 2,6-diaminopurine [225] , ϕisocytosine [226], E-base [227] , hypoxanthine [228], 2thiouracil [229], and 6-thioguanine [230], and each offered specific effects on the stability of the derived PNA-DNA hybrids. A basic requirement for triplex formation is that the central base of the triad must be able to form hydrogen bonds from both sides, and purine bases are ideal for this purpose [231].

A single incorporation of an amino-G-clamp was found to increase the Tm of a DNA decamer by 18 °C, presumably by the formation of four hydrogen bonds and enhanced stacking interactions. The amino-G clamp conferred enhanced potency to a 15-mer phosphorothioate (PS) antisense oligonucleotide that showed sequence specificity and RNase H activation [217]. Additionally, unaided cellular permeation was observed for a 7-mer PS-ODN incorporating the naked phenoxazine nucleobase analogueue (without the aminoethoxy arm) [218]. Furthermore, a single incorporation of an amino G-clamp at the 3’-end completely protects the oligonucleotides against 3’-exonuclease attack [219]. Corresponding PNA-DNA stability study is not done yet.

Cyanuric acid, a six-membered cyclic imide with alternate arrangement of hydrogen bond donors and acceptors is potentially well suited for such a purpose. In this context, Ganesh et al. [232] have presented a method for synthesis of cyanuryl-PNA monomer (2 3 2 ) that is useful in the preparation of new PNA analogueues (Fig. 51).

H 2N H2NCONHCONHHNO2

i

O

O

O HN

NH HN

O

ii

O

N

O

iv

O

N

O O

OR

OBn

228

NH

HN

NH

229

O

O

230. R = Benzyl

Boc

N H

N 232

iii 231. R = H Reagents and Conditions: (i) glycine benzyl ester toluene-4-sulfonate, Et 3N, DMF, 80 °C, 6 h; (ii) CDI, pyridine, reflux, 30 min; (iii) KOH, aqueou s MeOH, reflux, 45 min; (iv) eth yl N-(2-Boc-aminoethyl)-glycinate, HOBT, 0 °C, DCC, DMF.

Fig. (51). Synthesis of cyanuric acid PNA monomer.

COOEt

Peptide Nucleic Acids (PNAs)

Current Medicinal Chemistry, 2005, Vol. 12, No. 22

The structural features found in the crystal data of cyanuryl monomer (51) shows, conformational similarity to PNA oligomeric structures around bonds encompassing the tertiary amide group. The results indicate a preferred trans orientation of the side chain-carrying base in the monomer, identical to that found in the oligomeric PNA complexes. HN a

HN c2

b

B

g c1 N

d

O N

O

B

e O

O trans

cis

Fig. (52). Trans and cis orientation of the side chain-carrying base in the PNA monomer.

Until recently, three different modes of PNA binding to dsDNA (via duplex or triplex invasion and through formation of non-invasive triplex) have been described [233, 37]. However, they all have common sequence limitation: generally, one of the two dsDNA strands of the PNAbinding site must consist mostly of purines. Therefore, to

2593

expand the practical potential of PNA, an extension of the sequence repertoire for dsDNA recognition by PNAs is warranted. Pseudo Complementary PNAs One possible solution is the recent development of pseudo complementary PNAs (pcPNAs) carrying 2,6diaminopurine (D) and 2-thiouracil (sU) instead of adenine and thymine, respectively, along with ordinary guanine and cytosine (Fig. 53A and 53B) [234, 235]. These types of PNAs exhibits a distinct binding mode, double-duplex invasion, which is based on the Watson-Crick recognition principle supplemented by the notion of pseudocomplementarity. Pseudocomplementarity means that two special derivatives of initially paired normal purine and pyrimidine are structurally adjusted in such a way that they (i) do not match each other, but (ii) are capable of a stable Watson-Crick-type pairing with the natural nucleobase counterparts. The (D) and (sU) nucleobases were chosen for the design of the first pcPNAs, because they satisfied both these criteria [236, 237] (Fig. 53 B). The novel PNA variety may overcome sequence limitations of previously employed all pyrimidine PNA

HN

pcPNA C

A N

B

c1

B D *U C G

O

dsDNA

O pcPNA CH3

CH3 B

O

O H

N

H

H N

N

H

N

R

N

N

N

R

O

N

R

O

N

N

D

H

N

R

N N

N

N A

H

H N

R

H

N

S H

R T

O

O H

N

N

N

A

N H

N

T

H

H

U

N

N

N R

N D

H

N H

N

H

N S

R U

Strong interference

(A) Chemical s tructure of pcPNA. In pcPNA, the nucleobases A and T are substituted by the modified n ucleobases D and sU, respectiv ely. (B) The modified nucleobases prevent pcPNAs from forming a stablePNA-PNAduplex due to steric hindrance between D and sU bases, whereas they do not prevent pcPNAs from forming stable PNA-DNA heterod uplexes with complementary DNA strands. (C) Double-d uplex invasion complex formed at binding of a pair of pcPNAs to the target sequence in dsDNA.

Fig. (53). Pseudo complementary PNAs (pcPNAs) carrying 2,6-diaminopurine (D) and 2-thiouracil (sU) instead of adenine and thymine.

2594 Current Medicinal Chemistry, 2005, Vol. 12, No. 22

Porcheddu and Giacomelli

O 2N

O 2N

O 2N O

O

O NH

NH

NH N

O

HATU, DIEA

O

233

N

DBU

O

O O

O OH

Boc

N

N H

H N

O

O

O O

Boc

CN

233

N

N H

Boc

CN

O

N N H

234

OH 2 35, TNP

Fig. (54). Synthesis of t-butoxycarbonyl (Boc)-protected O4-(o-nitrophenyl) thymine peptide nucleic acid (PNA) monomers. openers that bind to oligopurine DNA sites only [238]. In accord with a strand-invasion mode of complex formation, the pcPNA binding proceeds much faster with super coiled than with linear plasmids. The double-duplex invasion complexes selectively shield specific DNA sites from BclI restriction endonuclease and dam methylase. The pcPNAassisted protection against enzymatic methylation is more efficient when the PNA-binding site embodies the methylase-recognition site rather than overlaps it. The site-selective conjugation of peptide nucleic acids (PNA) with fluorescent reporter groups is essential for the construction of hybridisation probes that can report the presence of a particular DNA sequence. PNA oligomers are good candidates as therapeutic agents, diagnostic tools and probes in molecular biology. For most of these applications, the functionalisation and labelling of PNA are necessary [239-242]. Labelled PNAs Labelled PNA can be prepared by modification of the Nterminal amino group [243-245]. This is the most common approach, but the reactive amino group can only be used for the introduction of one modification. Also trifunctional

amino acids such as cysteine or lysine or nucleobases carrying hexylamino groups have been introduced at various positions on the PNA oligomers to generate amino or thiol groups, which were further reacted with fluorescent labels [246, 247]. This method requires the previous synthesis of one monomer for each label. To avoid the need to synthesise a large number of different monomers, the reaction of fluorescent compounds to amino groups after the assembly of the oligomer was preferred. In the context, Eritja et al. [248] have decided the preparation of t-butoxycarbonyl (Boc)-protected O-4-(onitrophenyl) thymine peptide nucleic acid monomer (Fig. 54). PNA monomer (TNP) is a valuable intermediate for the introduction of fluorescent compounds or other compounds of interest in PNA oligomers, by nucleophilic displacement of the appropriate amino derivative. The post-synthetic modification of the oligomers to yield fluorescently labelled PNA oligomers was studied before and after the removal of the protecting groups. This method may be used to prepare several derivatives from the same PNA sequence. Also, this method may be used in combination with others [243, 249] to introduce multiple labels into the same

O 2N

O

HN N

R N

R-NH2 N

O

N

O

O

O

236

237

NH(CH3) 2

HO

O

O

R-NH2 = COOH SO2NH(CH2) 5NH2 NHCSNH(CH2) 5NH3Br

Fig. (55). Labelled PNA monomers by a post-synthetic modification approach.

Peptide Nucleic Acids (PNAs)

Current Medicinal Chemistry, 2005, Vol. 12, No. 22

2595

compound. For example, when monomer (TNP) is placed at any position, the amino terminal group is still free to react with a second label (Fig. 55). The introduction of two different fluorescent labels in PNA is of interest for the preparation of molecular beacons [247, 250, 251], for diagnosis and biomedical studies [252].

Cbz

= Benzyloxycarbonyl

CD

= Circular Dichroism

CDI

= N’,N’-Carbonyldiimidazole

CeNA

= Cyclohexenyl nucleic acid

ChPNA

= Cyclohexane PNA

CONCLUDING REMARKS

Cyt

= Cytosine

As this review hopefully has demonstrated, numerous variations over the PNA structure are feasible, and many have been realised. Some recent efforts to provide further applications of this exciting nucleic acid analogue include modifications of backbone and the development of novel base analogue. Studies on these new chemical modifications of the original PNA backbone could contribute to increasing the potentialities of PNAs and lead to the development of novel applications and PNA-dependent projects in many areas of biology and therapy.

DBU

= 1,8-Diazabicyclo[5.4.0]undec-7-ene

DCA

= Dichloroacetic acid

DCC

= N'-N'-Dicyclohexylamide

DCE

= 1,2-Dichloroethane

DCM

= Dichloromethane

Dde

= 1-(4,4-Dimethyl-2, 6-dioxacyclohexylidene) thyl

DIEA=

= Diisopropylethylamine

In brief, the application of PNA as a gene-targeted therapeutic agent still has to await the development of efficient methods for its uptake and cell penetration. By contrast, its application as a diagnostic means has already led to promising developments in many areas of chemistry, biology, and biotechnology.

DIPEA DMA

= Dimethylacetamide

DMAP

= 4-Dimethylaminopyridine

DME

= 1,2-Dimethoxyethane

DMF

= N,N-Dimethylformamide

ACKNOWLEDGEMENT

DMSO

= Dimethyl sulfoxide

The authors thank the reviewer for his helpful discussions and critical readings of the manuscript. The work was financially supported by the University of Sassari and MIUR (Rome) within the project PRIN 2003.

DMT

= Dimethoxytrityl

DNA

= Deoxyribonucleic acid

dsDNA

= Double-stranded DNA

ssDNA

= Single-stranded DNA

LIST OF ABBREVIATIONS

EDTA

= Ethylenediaminetetraacetic acid

A

= Adenine

EDC

Aba

= 4-Aminobutyric acid

= 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide

Ac

= Acetyl

Fmoc

= 9-Fluorenylmethoxycarbonyl

Acm

= Acetamidomethyl

G

= Guanine

Gly

= Glycine

Gua

= Guanine

HATU

= O-(7-Azabenzotriazol-1-yl)-N,N,N’,N’-tetrametyluronium hexafluorophosphate

HBTU

= O-(Benzotriazol-1-yl)-N,N,N’,N’-tetramethyluronium hexafluorophosphate

ASGB-R = Hepatic asialoglycoprotein receptor

HMBA

= 4-(Hydroxymethyl)benzoic acid

Base

= Heterocyclic nucleobase

HNA

= Hexitol Nucleic Acids

Bhoc

= Benzhydryloxycarbonyl

HOBT

= 1-Hydroxybenxotriazole

Bipy

= 2,2’-Bipyridyl

HPLC

= High pressure liquid chromatography

Boc

= t-Butoxycarbonyl

LNA

= Locked nucleic acid

Bz

= Benzoyl

LTR

= Long terminal repeat

Bzl

= Benzyl

Lys

= Lysine

C

= Cytosine

MMT

= Monomethoxytrityl

CAN

= Cyclohexanyl nucleic acid

MsCl

= Mesyl

aegPNA = Aminoethylglycine peptide nucleic acid Ala

= Alanine

All

= Allyl

Alloc

= Allyloxycarbonyl

APNA

= Aromatic peptide nucleic acids

2596 Current Medicinal Chemistry, 2005, Vol. 12, No. 22

Porcheddu and Giacomelli

NADI

= Naphthalene diimide

[3]

NEM

= N-Ethylmaleimide

NMP

= N-Methyl-2-pyrrolidinone

[4] [5] [6]

NMR

= Nuclear Magnetic Resonance

[7]

o-Ns-Cl

= O-Nitrobenzenesulfonyl chloride

OPA

= Olefin Polyamide Nucleic Acid

PAM

= Pyridoxamine resin

[9] [10]

PANA

= Polyamine nucleic acid

[11]

PCR

= Polymerase chain reaction

PEGA

= Acryloylated O, O’-bis(2-aminopropyl) polyethylene glycol resin

pcPNA

= Pseudo complementary peptide nucleic acid

Phe

= Phenylalanine

PG

= Protective group

p-RNA

= Pyranosyl ribonucleic acid

PyBop

= Benzotriazole-1-yl-oxy-tris-pyrrolidinophos-

[8]

[12] [13] [14] [15] [16] [17] [18]

phonium hexafluorophosphate [19] [20]

PNA

= Peptide nucleic acid

PS

= Phosphorothioate

RNA

= Ribonucleic acid

Ser

= Serine

SSTRs

= Somatostatin-receptors

[23]

T

= Thymine

[24]

Tm

= Thermal melting

TFA

= Trifluoroacetic acid

[21] [22]

[25]

TFMSA = Trifluoromethanesulfonic acid [26]

TAR

= Transactivation response element

TBTP

= Thiobutyltriphenylphosphonium bromide

[27]

TFA

= Trifluoroacetic acid

[28]

THF

= Tetrahydrofuran

Thr

= Threonine

Trp

= Tryptophan

TNA

= Threofuranosyl nucleic acids

Tyr

= Tyrosine

TPP

= Triphenylphosphonium

Trt

= Trityl

tcypPNA = trans-Cyclopentane peptide nucleic acid TNA

= Threofuranosyl nucleic acids

Ts

= 4-Methylbenzensulfonyl

UGI-4CC = UGI 4 Components condensation reactions

[29] [30] [31] [32] [33] [34]

REFERENCES [35] [1] [2]

Nielsen, P. E. Bioconjug. Chem. 1991, 2, 1. Dervan, P. B. Bioorg. Med. Chem. 2001, 9, 2215.

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