BACKGROUND OF THE INVENTION From the discovery of penicillin in the 1940's there has been an ever-growing search for new drugs. Many drugs or antibiotics have been discovered or developed from already existing drugs. However, over the years many strains of bacteria have become resistant to one or more of the currently available drugs, which were effective, drugs in the past. The number of antibiotic drugs currently being used by clinicians is more than 100.

Most antibiotics are products of natural microbic populations and resistant traits found in these populations can disseminate between species and appear to have been acquired by pathogens under selective pressure from antibiotics used in agriculture and medicine (Davies 1994 (1)). Antibiotic resistance may be generated in bacteria harbouring genes that encode enzymes that either chemically alter or degrade the antibiotics. Another possibility is that the bacteria encodes enzymes that makes the cell wall impervious to antibiotics or encode efflux pumps that eject antibiotics from the cells before they can exert their effects.

Because of the emergence of antibiotic resistant bacterial pathogens, there is an on-going need for new therapeutic strategies. One strategy to avoid problems caused by resistance genes is to develop anti-infective drugs from novel chemical classes for which specific resis- tance traits do not exist.

Antisense agents offer a novel strategy in combating diseases, as well as opportunities to employ new chemical classes in the drug design.

Oligonucleotides can interact with native DNA and RNA in several ways. One of these is du- plex formation between an oligonucleotide and a single stranded nucleic acid. Another is tri- plex formation between an oligonucleotide and double stranded DNA to form a triplex struc-

ture.

Results from basic research have been encouraging, and antisense oligonucleotide drug formulations against viral and disease causing human genes are progressing through clinical trials. Efficient antisense inhibition of bacterial genes also could have wide applications; however, there have been few attempts to extend antisense technology to bacteria.

Peptide nucleic acids (PNA) are compounds that in certain respects are similar to oligonu- cleotides and their analogs and thus may mimic DNA and RNA. In PNA, the deoxyribose backbone of oligonucleotides has been replaced by a pseudo-peptide backbone (Nielsen et al. 1991 (2)) (Fig. 1). Each subunit, or monomer, has a naturally occurring or non-naturally occurring nucleobase attached to this backbone. One such backbone is constructed of re- peating units of N- (2-aminoethyl) glycine linked through amide bonds. PNA hybridises with complementary nucleic acids through Watson and Crick base pairing and helix formation (Egholm et al. 1993 (3)). The Pseudo-peptide backbone provides superior hybridization prop- erties (Egholm et al. 1993), resistance to enzymatic degradation (Demidov et al. 1994 (4)) and access to a variety of chemical modifications (Nielsen and Haaima 1997 (5)).

PNA binds both DNA and RNA to form PNA/DNA or PNA/RNA duplexes. The resulting PNA/DNA or PNA/RNA duplexes are bound with greater affinity than corresponding DNA/DNA or DNA/RNA duplexes as determined by Tm's. This high thermal stability might be attributed to the lack of charge repulsion due to the neutral backbone in PNA. In addition to increased affinity, PNA has also been shown to bind to DNA with increased specificity. When a PNA/DNA duplex mismatch is melted relative to the DNA/DNA duplex, there is seen an 8 to 20°C drop in the Tm.

Furthermore, homopyrimidine PNA oligomers form extremely stable PNA2-DNA triplex with sequence complementary targets in DNA or RNA oligomers. Finally, PNA's may bind to dou- ble stranded DNA or RNA by helix invasion.

An advantage of PNA compared to oligonucleotides is that the PNA polyamide backbone (having appropriate nucleobases or other side chain groups attached thereto) is not recog- nised by either nucleases or proteases and are thus not cleaved. As a result, PNA's are re- sistant to degradation by enzymes unlike nucleic acids and peptides.

For antisense application, target bound PNA can cause steric hindrance of DNA and RNA polymerases, reverse transcription, telomerase and the ribosome's (Hanvey et al. 1992 (6), Knudsen et al. 1996 (7), Good and Nielsen 1998 (12)), etc.

A general difficulty when using antisense agents is cell uptake. A variety of strategies to im- prove uptake can be envisioned and there are reports of improved uptake into eukaryotic cells using lipids (Lewis et al. 1996 (8)), encapsulation (Meyer et al. 1998 (9)) and carrier strategies (Nyce and Metzger 1997 (10), Pooga et al, 1998 (11)).

WO 99/05302 discloses a PNA conjugate consisting of PNA and the transporter peptide transportan, which peptide may be used for transport cross a lipid membrane and for delivery of the PNA into interactive contact with intracellular polynucleotides.

US-A-5 777 078 discloses a pore-forming compound which comprises a delivery agent rec- ognising the target cell and being linked to a pore-forming agent, such as a bacterial exotoxin. The compound is administered together with a drug such as PNA.

As an antisense agent for microorganisms, PNA may have unique advantages. It has been demonstrated that PNA based antisense agents for bacterial application can control cell growth and growth phenotypes when targeted to Escherichia coli rRNA and mRNA (Good and Nielsen 1998a, b (12,13), (and WO 99/13893).

However, none of these disclosures discuss ways of transporting the PNA across the bacte- rial cell wall and membrane.

Furthermore, for bacterial application, poor uptake is expected, because bacteria have strin- gent barriers against foreign molecules and antisense oligomer containing nucleobases ap- pear to be too large for efficient uptake. The results obtained by Good and Nielsen (1998a, b (12,13)) indicate that PNA oligomers enter bacterial cells poorly by passive diffusion across the lipid bilayer.

US-A-5 834 430 discloses the use of potentiating agents, such as short cationic peptides in the potentiation of antibiotics. The agent and the antibiotic are co-administered.

WO 96/11205 discloses PNA conjugates, wherein a conjugated moiety may be placed on

terminal or non terminal parts of the backbone of PNA in order to functionalise the PNA. The conjugated moieties may be reporter enzymes or molecules, steroids, carbohydrate, ter- penes, peptides, proteins, etc. It is suggested that the conjugates among other properties may possess improved transfer properties for crossing cellular membranes. However, WO 96/11205 does not disclose conjugates, which may cross bacterial membranes.

WO 98/52614 discloses a method of enhancing transport over biological membranes, e. g. a bacterial cell wall. According to this publication, biological active agents such as PNA may be conjugated to a transporter polymer in order to enhance the transmembrane transport. The transporter polymer consists of 6-25 subunits; at least 50% of which contain a guanidino or amidino sidechain moiety and wherein at least 6 contiguous subunits contain guanidino and/or amidino sidechains. A preferred transporter polymer is a polypeptide containing 9 ar- ginine.

WO 98/03542 discloses Peptide nucleic acids having enhanced binding affinity including PNAs with amino acid side chain modifications.

Antisense PNAs as antibiotics How to design an antisense PNA has recently been reviewed (Knudsen et al. 1996 (7), Good and Nielsen, 1997 (16)). The first report about PNA as antibi- otics appeared in March 1998 (Good and Nielsen 1998a (12)) Translation was inhibited by targeting PNAs to 23S rRNA of the ribosome in E. Coli. A triplex forming bis PNA (H- JTJTJJT- (eg1) 3-TCCTCTC-LysNH2) complementary to a 7-mer homopurine sequence in the alpha-sarcin loop of 23S rRNA inhibited translation at 100 nM concentration in vitro, and of 2 tM concentration in vivo. A more permeable strain, E. Coli AS19 were 10 times as sensitive towards the PNA as wildtyp E. Coli K-12, indicating that uptake was responsible for the modest (but significant) antibacterial effect. Similar a triplex forming bis-PNA targeting the almost single stranded peptidyl transferase center showed almost the same effect. Duplex forming PNAs (targeting the peptidyl transferase center or the mRNA binding region of 16S rRNA) did not show any effect below 0.5 pLM in vitro, and below 20 M concentration in vivo.

The alpha-sarcin loop contains the longest universally conserved sequence (12 nt) of all rRNA and is therefore not a good target for an antibacterial PNA (Meyer et al. 1996 (17)).

The peptidyl transferase center is a possible target though.

Almost at the same time the same authors published the Nature Biotechnology paper:"An- tisense inhibition of gene expression in bacteria by PNA targeted to mRNA" (Good and Niel- sen 1998b (13)). Part of this work has also been published as a protocol (Good and Nielsen,

1999 (18)). This time PNAs were designed to target the start codon regions of the E. Coli p- galactosidase and ß-lactamase genes. In both cases dose dependent and specific gene inhi- bition was observed in vitro using nM concentrations and in vivo using M concentrations.

The last target will be treated in some details since this is relevant to the work described be- low. The ß-lactamase gene codes for the ß-lactamase enzyme which cleaves the antibiotic pennicilin (which contains a four-membered lactam ring). This gene is not part of the E. Coli chromosome. Instead it is a plasmid (a small piece of circular dsDNA) taken up by the E. Coli from the outside. One way of killing resistant bacteria could be to inhibit protein synthesis of the ß-lactamase enzyme with an antisense PNA. The completed nucleotide sequence of the P-lactamase gene was published in 1978 (Figure 6) (Sutcliffe, 1978 (19)) This gene codes for a protein of 286 amino acids. By reading the coding strand, the following appears: The E. Coli RNA polymerase binds at the promotor sequence containing the so- called Pribnow box (indicated) and transcribes the gene to a mRNA copy. The ribosome starts the protein synthesis from the mRNA at the start codon ATG (indicated). This area is the best place to target the duplex forming antisense PNA. The 12-mer PNA sequence used in this study is underlined. In the Nature Biotechnology paper (Good and Nielsen 1998b (13)) two very similar PNAs were used, both three residues longer: PNA 1438 was a 15 mer with 3 more residues in the C-terminal part, and PNA 1439 had 3 more residues in the N terminal part of the PNA used in this study. The first 23 amino acids are believed to be a secretion signal since this hydrophobic peptide is not part of the mature enzyme. Strangely, no triplex forming bis PNAs were targeted against any of the three 7 nt homo purine targets available in the mRNA (indicated in the coding strand).

SUMMARY OF THE INVENTION At present, the main problem in using antisense agents to treat bacterial infections is their poor uptake. The present invention relates to modifications of the PNAs improving the uptake of the PNAs.

It has previously been shown that antisense PNA can inhibit growth of bacteria. However, due to a slow diffusion of the PNA over the bacterial cell wall a practical application of the PNA as an antibiotic has not been possible previously. According to the present invention, a practical application in tolerable concentration may be achieved by modifying the PNA by linking a peptide or peptide-like sequence, which enhances the activity of the PNA.

Surprisingly, it has been found out that by incorporating a peptide, an enhanced anti-infective effect can be observed. The important feature of the modified PNA molecules seems to be a pattern comprising in particular positively charged and lipophilic amino acids or amino acid analogues. An anti-infective effect is found with different orientation of the peptide in relation to the PNA-sequence.

Thus, the present invention concerns a PNA monomer of formula (ll) : <BR> (II) wherein B is a naturally-occurring nucleobase or a non-naturally-occurring nucleobase ; (Pr) is hydrogen or a protection group; and R is C, 6-alkyl, 3-guanidinopropyl, carboxymethyl, aminocarboxymethyl, mercaptomethyl, 2-carboxyethyl, aminocarboxyethyl, imidazol-4-yl-methyl,, 4-aminobutyl, 2- (methylthio) ethyl, benzyl, hydroxymethyl, 1-hydroxyethyl, 3-indolyl, 4-hydroxybenzyl, 2-hydroxymethyl, 3- ureidopropyl or 4-pyridomethyl ; and a PNA oligomer with from 6 to 25 monomers selected from the group consisting of aeg- PNA monomer and a monomer of formula (II) and a modified PNA molecule of formula (I) Q-L-PNA (I) wherein L is a linker or a bond; Q is a peptide and PNA is a peptide nucleic acid oligomer with from 6 to 25 monomers selected from the group consisting of aeg-PNA monomer and a monomer of formula (II).

The protection group is selected from, Boc, (tert-butyloxycarbonyl), Cbz (benzyloxycarbonyl), Fmoc (fluorenylmethyloxycarbonyl), Mmt (monomethoxytrityl) or another group known by those skilled in the art.

In another aspect of the invention the modified PNA molecules are used in the manufacture of medicaments for the treatment or prevention of a disease selected from bacterial and viral infections, cardiac or vascular diseases, metabolic diseases or immunological disorders or for disinfecting non-living objects.

In a further aspect, the invention concerns a composition for treating or preventing infectious diseases or disinfecting non-living objects.

In yet another aspect, the invention concerns the treatment or prevention of infectious dis- eases or treatment of non-living objects.

In yet a further aspect, the present invention concerns a method of identifying specific advan- tageous antisense PNA sequences, which may be used in the modified PNA molecule ac- cording to the invention.

BRIEF DESCRIPTION OF THE DRAWINGS FIGURE 1 shows the chemical structure of DNA and PNA oligomers. The PNA oligomer has a backbone constructed of repeating units of N- (2-aminoethyl) glycine linked through amide bonds. The repeating unit is designated"aeg". Each monomer unit designated"aeg-PNA monomer"is also shown.

FIGURE 2 shows the principle in conjugation using SMCC FIGURE 3 shows the nucleotide sequence of the mrcA (ponA) gene encoding PBP1A. The sequence of the gene (accession number X02164) was obtained from the EMBL sequence database (Heidelberg, Germany) (Broome-Smith et al. 1985, Eur J Biochem 147 : 437-46 (14)). Two possible start codons have been identified (highlighted). Bases 1-2688 are shown (ending with stop codon).

FIGURE 4 shows the nucleotide sequence of the mrdA gene encoding PBP2. The sequence (accession number AE000168, bases 4051-5952, numbered 1-2000) was obtained from the E. coli genome database at the NCBI (Genbank, National Centre for Biotechnology Informa-

tion, USA). The start codon is highlighted.

FIGURE 5 shows the chemical structures of the different succinimidyl based linking groups used in the conjugation of the Peptide and PNA FIGURE 6 shows the sequence of the R-Lactamase gene. The top strand represents the coding strand (5'-3') and the bottom strand represents the template strand (3'-5').

FIGURE 7 Bar graph showing the anti-Lactamase activity of the PNA's (Table 1) in E. Coli K12 at 1 FM conc.

FIGURE 8 Bar graph showing the anti ß-Lactamase activity of the PNA's (Table 1) in E. Coli K12 at 0. 5 IlM conc.

FIGURE 9 Bar graph showing the anti-Lactamase activity of the PNA's (Table 1) in E. Coli AS19 at 1 M conc.

FIGURE 10 Bar graph showing the anti 3-Lactamase activity of the PNA's (Table 1) in E. Coli AS19 at 0. 5 zM conc.

FIGURE 11: Graph showing the antisense effect of normal aminoethylglycine PNA (1833) compared to the corresponding PNA (1883) in which three positions (TLys) have been re- placed by a lysine-T PNA monomer (R being 4-aminobutyl). The PNAs were targeted to the translation initiation region of the E. coli beta-galactosidase gene (lacZ). Cultures of E. coli were incubated with the indicated concentrations of PNA, and the beta-galactosidase activity was measured after overnight growth.

DETAILED DESCRIPTION OF THE INVENTION Antisense PNA's can inhibit bacterial gene expression with gene and sequence specificity (Good and Nielsen 1998a, b (12,13) and WO 99/13893). The approach may prove practical as a tool for functional genomics and as a source for novel antimicrobial drugs. However, im- provements on standard PNA are required to increase antisense potencies. The major limit to activity appears to be cellular entry. Bacteria effectively exclude the entry of large molecu-

lar weight foreign compounds, and previous results for in vitro and cellular assays seem to show that the cell barrier restricts antisense effects. Accordingly, the present invention con- cerns strategies to improve the activity of antisense potencies.

Without being bound by theory, it is believed that the short cationic peptides lead to an im- proved PNA uptake over the bacterial cell wall. It is believed that the short peptides act by penetrating the cell wall, allowing the modified PNA molecule to cross the cell wall to get ac- cess to structures inside the cell, such as the genome, mRNA's, the ribosome, etc. However, an improved accessibility to the nucleic acid target or an improved binding of the PNA may also add to the overall effect observed.

According to the invention, PNA molecules modified with short activity enhancing peptides enable specific and efficient inhibition of bacterial genes with nanomolar concentrations. An- tisense potencies in this concentration are consistent with practical applications of the tech- nology. It is believed that the present invention for the first time demonstrates that peptides with a certain pattern of cationic and lipophilic amino acids can be used as carriers to deliver agents and other compounds into micro-organisms, such as bacteria. Further, the present invention has made it possible to administer PNA in an efficient concentration, which is also acceptable to the patient.

The terms"Ct 6-alkyl"as used herein, represent a branched or straight alkyl group having from one to six carbon atoms. Typical C, 6-alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, iso-propyl, butyl, iso-butyl, sec-butyl, tert-butyl, pentyl, iso-pentyl, hexyl, iso- hexyl and the like.

By the terms"cationic amino acids and amino acid analogues"and"positively charged amino acids and amino acid analogues"are to be understood any natural or non-natural occurring amino acid or amino acid analogue which have a positive charge at physiological pH. Simi- larly the term"non-charged amino acids or amino acid analogs"is to be understood any natural or non-natural occurring amino acids or amino acid analogs which have no charge at physiological pH.

Among the positively charged amino acids and amino acid analogs may be mentioned lysine (Lys, K), arginine (Arg, R), diamino butyric acid (DAB) and ornithine (Orn). The skilled person will be aware of further positively charged amino acids and amino acid analogs.

Among the non-charged amino acids and amino acid analogs may be mentioned the natural occurring amino acids alanine (Ala, A), valine (Val, V), leucine (Leu, L), isoleucine (Ile, I), proline (Pro, P), phenylanaline (Phe, F), tryptophan (Trp, W), methionin (Met, M), glycine (Gly, G), serine (Ser, S), threonine (Thr, T), cysteine (Cys, C), tyrosine (Tyr, Y), asparagine (Asn, N) and glutamine (Gln, Q), the non-natural occurring amino acids 2-aminobutyric acid, ß-cyclohexylalanine, 4-chlorophenylalanine, norleucine and phenylglycine. The skilled person will be aware of further non-charged amino acids and amino acid analogs.

Preferably, the non-charged amino acids and amino acid analogs are selected from the natu- ral occurring non-polar amino acids Ala, Val, Leu, Ile, Phe, Trp and Met or the non-natural occurring non-polar amino acids ß-cyclohexylalanine, 4-chlorophenylalanine and norleucine.

The term"functionally similar moiety"is intended to cover all peptide-like molecules which functionally mimic the Peptide as defined above and thus impart to the PNA molecule the same advantageous properties as the peptides comprising natural and non-natural amino acids as defined above.

Examples of preferred modified PNA molecules according to the invention are (Lys Phe Phe) 3 Lys-L-PNA and any subunits thereof comprising at least three amino acids. One pre- ferred Peptide is (Lys Phe Phe) 3. Others are (Lys Phe Phe) 2 Lys Phe, (Lys Phe Phe) 2 Lys, (Lys Phe Phe) 2, Lys Phe Phe Lys Phe, Lys Phe Phe Lys and Lys Phe Phe as disclosed in PCT Publication WO 01/27261.

The number of amino acids in the peptide may be chosen between 3 and 20. It appears that at least 3 amino acids; whereof at least one is a positively charged amino acid is necessary to obtain the advantageous effect. On the other hand, the upper limit only seems to be lim- ited by an upper limit of the overall size of the PNA molecule for the purpose of the practical use of said molecule. Preferably, the total number of amino acids is 15 or less, more prefer- able 12 or less and most preferable 10 or less.

The PNA molecule is connected to the Peptide moiety through a direct binding or through a linker. A variety of linking groups can be used to connect the PNA with the Peptide.

Linking groups are described in WO 96/11205, WO 01/27261 and W098/52614, the content of which are hereby incorporated by reference.

Some linking groups may be advantageous in connection with specific combinations of PNA and Peptide.

Preferred linking groups are ADO (8-amino-3,6-dioxaoctanoic acid), SMCC (succinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate) AHEX or AHA (6-aminohexanoic acid), 4- aminobutyric acid, 4-aminocyclohexylcarboxylic acid, LCSMCC (succinimidyl 4- (N- maleimidomethyl) cyclohexane-1-carboxy-(6-amido-caproate), MBS (succinimidyl m- maleimido-benzoylate), EMCS (succinimidyl N-E-maleimido-caproylate), SMPH (succinimidyl 6-(ß-maleimido-propionamido) hexanoate, AMAS (succinimidyl N- (a-maleimido acetate), SMPB (succinimidyl 4- (p- maleimidophenyl) butyrate), P. ALA (ß-alanine), PHG (Phenylgly- cine), ACHC (4-aminocyclohexanoic acid), R. CYPR (3- (cyclopropyl) alanine), ADC (amino dodecanoic acid), polyethylene glycol's and amino acids.

Any of these groups may be used as a single linking group or together with more groups in creating a suitable linker. Further, the different linking groups may be combined in any order and number in order to obtain different functionalities in the linker arm.

In the case SMCC (succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate) is used in the process of linking PNA to the peptide, it is necessary to add a cysteine (C) or a similar thiol containing moiety to the terminal end of the peptide (see Fig. 2). Additionally, amino ac- ids, such as glycine, may be a part of the linker.

The chemical structures of the different succinimidyl based linking groups used in the conju- gation of the Peptide and PNA is shown in Figure 5.

The Peptide is normally linked to the PNA sequence via the amino or carboxy end. However, the PNA sequence may also be linked to an internal part of the peptide.

The modified PNA molecule according to the present invention comprises a PNA oligomer of a sequence, which is complementary to at least one target nucleotide sequence in a micro- organism, such as a bacterium. The target may be a nucleotide sequence of any RNA, which is essential for the growth, and/or reproduction of the bacteria. Alternatively, the target may be a gene encoding a factor responsible for resistance to antibiotics, In a preferred embodi- ment, the functioning of the target nucleotide sequence is essential for the survival of the bacteria and the functioning of the target nucleic acid is blocked by the PNA sequence, in an antisense manner.

The binding of a PNA strand to a DNA or RNA strand can occur in one of two orientations, anti-parallel or parallel. As used in the present invention, the term complementary as applied to PNA does not in itself specify the orientation parallel or anti-parallel. It is significant that the most stable orientation of PNA/DNA and PNA/RNA is anti-parallel. In a preferred em- bodiment, PNA targeted to single strand RNA is complementary in an anti-parallel orienta- tion.

In a another preferred embodiment of the invention a bis-PNA consisting of two PNA oli- gomers covalently linked to each other is targeted to a homopurine sequence (consisting of only adenine and/or guanine nucleotides) in RNA (or DNA), with which it can form a PNAs- RNA (PNA2-DNA) triple helix.

In another preferred embodiment of the invention, the PNA contains from 5 to 20 nucleo- bases, in particular from 7-15 nucleobases, and most particular from 8 to 12 nucleobases.

Peptide Nucleic Acids are described in WO 92/20702 and WO 92/20703, the content of which is hereby incorporated by reference.

Potential target genes may be chosen based on the knowledge about bacterial physiology. A target gene may be found among those involved in one of the four major process complexes : cell division, cell wall synthesis, protein synthesis (translation) and nucleic acid synthesis. A target gene may also be involved in antibiotic resistance.

A further consideration is that some physiological processes are primarily active in dividing cells whereas others are running under non-dividing circumstances as well.

Known target proteins in cell wall biosynthesis are penicillin binding proteins, PBPs, the tar- gets of, e. g., the beta-lactam antibiotic penicillin. They are involved in the final stages of cross-linking of the murein sacculus.

E. coli has 12 PBPs, the high molecularweight PBPs : PBP1a, PBP1b, PBP1c, PBP2 and PBP3, and seven low molecular weight PBPs, PBP 4-7, DacD, AmpC and AmpH. Only the high molecular weight PBPs are known to be essential for growth and have therefore been chosen as targets for PNA antisense.

Protein biosynthesis is an important process throughout the bacterial cell cycle. Therefore, the effect of targeting areas in the field of protein biosynthesis is not dependent on cell divi- sion.

Both DNA and RNA synthesis are target fields for antibiotics. A known target protein in DNA synthesis is gyrase. Gyrase acts in replication, transcription, repair and restriction. The en- zyme consists of two subunits, both of which are candidate targets for PNA.

Examples of potential targets primarily activated in dividing cells are rpoD, gyrA, gyrB, (tran- scription), mrcA (ponA), mrcB (ponB, pbpF), mrdA, ftsl (pbpB) (Cell wall biosynthesis), ftsQ, ftsA and ftsZ (cell division).

Examples of potential targets also activated in non-dividing cells are inM, infB, infC, tufAltufB, tsf, fusA, prfA, prfB, and prfC, (Translation).

Other potential target genes are antibiotic resistance-genes. The skilled person would readily know from which genes to choose. Two examples are genes coding for beta-lactamases in- activating beta-lactam antibiotics, and genes encoding chloramphenicol acetyl transferase.

PNA's against such resistance genes could be used against resistant bacteria.

Infectious diseases are caused by micro-organisms belonging to a very wide range of bacte- ria, viruses, protozoa, worms and arthropods and from a theoretical point of view PNA can be modified and used against all kinds of RNA in such micro-organisms, sensitive or resistant to antibiotics.

Examples of micro-organisms which may be treated in accordance with the present invention are Gram-positive organisms such as Streptococcus, Staphylococcus, Peptococcus, Bacil- lus, Listeria, Clostridium, Propionebacteria, Gram-negative bacteria such as Bacteroides, Fusobacterium, Escherichia, Klebsiella, Salmonella, Shigella, Proteus, Pseudomonas, Vibrio, Legionella, Haemophilus, Bordetella, Brucella, Campylobacter, Neisseria, Branhamella, and organisms which stain poorly or not at all with Gram's stain such as Mycobacteria, Tre- ponema, Leptospira, Borrelia, Mycoplasma, Clamydia, Rickettsia and Coxiella,

The ability of PNA's to inhibit bacterial growth may be measured in many ways, which should be clear to the skilled person. For the purpose of exemplifying the present invention, the bac- terial growth is measured by the use of a microdilution broth method according to NCCLS guidelines. The present invention is not limited to this way of detecting inhibition of bacterial growth.

To illustrate one example of measuring growth and growth inhibition the following procedure may be used: Bacterial strain: E. coli K12 MG1655 Media: 10% Mueller-Hinton broth, diluted with sterile water.

10% LB broth diluted with sterile water.

100% Mueller-Hinton broth.

Trays : 96 well trays, Costar # 3474, Biotech Line AS, Copenhagen. (Extra low sorbent trays are used in order to prevent/minimize adhesion of PNA to tray surface).

A logphase culture of E. coli is diluted with fresh preheated medium and adjusted to defined OD (here: Optical Density at 600 nm) in order to give a final concentration of 5x105 and 5x104 bacteria/ml medium in each well, containing 200 ll of bacterial culture. PNA is added to the bacterial culture in the wells in order to give final concentrations ranging from 300 nM to 1000 nM. Trays are incubated at 37°C by shaking in a robot analyzer, PowerWavex, soft- ware KC4 Kebo. Lab, Copenhagen, for 16 h and optical densities are measured at 600 nM during the incubation time in order to record growth curves. Wells containing bacterial culture without PNA are used as controls to ensure correct inoculum size and bacterial growth dur- ing the incubation. Cultures are tested in order to detect contamination.

The individual peptide-L-PNA constructs have MW between approx. 4200 and 5000 depend- ing on the composition. Therefore all tests were performed on a molar basis rather than on a weight/volume basis. However, assuming an average MW of the construct of 4500 a concen- tration of 500 nM equals 2.25 microgram/ml.

Growth inhibitory effect of PNA-constructs: The bacterial growth in the wells is characterized by the following phases: lag phase i. e. the period until growth starts,

log phase i. e. the period with maximal growth rate, steady-state phase followed by death phase.

These parameters are used evaluating the inhibitory effect of the PNA constructs on the bac- terial growth by comparing the growth curves of samples with and without PNA. constructs added.

Total inhibition of bacterial growth is defined as: OD (16h) = OD (Oh) or no visible growth ac- cording to NCCLS Guidelines In an initial screening the modified PNA molecules are tested in the sensitive 10% medium assay. Positive results are then run in the 100% medium assay in order to verify the inhibi- tory effect in a more"real"environment (cf. the American guidelines (NCCLS)).

In vivo antibacterial efficacy is established by testing a compound of the invention in the mouse peritonitis/sepsis model as described by N. Frimodt-Moller et al. 1999, Chap. 14, Handbook of Animal Models of Infection.

For the in vivo efficacy experiment a number of female NMRI mice are inoculated with ap- proximately 107 cfu of E. coli ATCC 25922 intraperitoneally. Samples are drawn from blood and peritoneal fluid at 1,2,4 and 6 hrs post infection, and cfu/ml counted. 1 hr post infection the animals are treated once in groups with: 1. Gentamicin (38 mg/kg s. c.); 2. Ampicillin (550 mg/kg s. c.); 3. a compound of the invention (50-60 mg/kg i. v.); 4. no treatment.

In another aspect of the present invention, the modified PNA molecules can be used to iden- tify preferred targets for the PNA. Based upon the known or partly known genome of the tar- get micro-organisms, e. g. from genome sequencing or cDNA libraries, different PNA se- quences can be constructed and linked to an effective anti-infective enhancing Peptide and thereafter tested for its anti-infective activity. It may be advantageous to select PNA se- quences shared by as many micro-organisms as possible or shared by a distinct subset of micro-organisms, such as for example Gram-negative or Gram-positive bacteria, or shared by selected distinct micro-organisms or specific for a single micro-organism.

In a further aspect of the present invention, the invention provides a composition for use in inhibiting growth or reproduction of infectious micro-organisms comprising a modified PNA

molecule according to the present invention. In one embodiment, the inhibition of the growth of micro-organisms is obtained through treatment with either the modified PNA molecule alone or in combination with antibiotics or other anti-infective agents. In another embodiment, the composition comprises two or more different modified PNA molecules. A second modi- fied PNA molecule can be used to target the same bacteria as the first modified PNA mole- cule or in order to target different bacteria. In the latter form, specific combinations of target bacteria may be selected to the treatment. Alternatively, the target can be one or more genes, which confer resistance to one or more antibiotics to one or more bacteria. In such a treatment, the composition or the treatment further comprises the use of said antibiotic (s).

In another aspect, the present invention includes within its scope pharmaceutical composi- tions comprising, as an active ingredient, at least one of the compounds of the general for- mula I or a pharmaceutical acceptable salt thereof together with a pharmaceutical ac- ceptable carrier or diluent.

Pharmaceutical compositions containing a compound of the present invention may be pre- pared by conventional techniques, e. g. as described in Remington: The Science and Practise of Pharmacy, 1 gth Ed., 1995. The compositions may appear in conventional forms, for exam- ple capsules, tablets, aerosols, solutions, suspensions or topical applications.

Typical compositions include a compound of formula I or a pharmaceutical acceptable acid addition salt thereof, associated with a pharmaceutical acceptable excipient which may be a carrier or a diluent or be diluted by a carrier, or enclosed within a carrier which can be in the form of a capsule, sachet, paper or other container. In making the compositions, conven- tional techniques for the preparation of pharmaceutical compositions may be used. For ex- ample, the active compound will usually be mixed with a carrier, or diluted by a carrier, or en- closed within a carrier which may be in the form of a ampoule, capsule, sachet, paper, or other container. When the carrier serves as a diluent, it may be solid, semi-solid, or liquid material which acts as a vehicle, excipient, or medium for the active compound. The active compound can be adsorbed on a granular solid container for example in a sachet. Some ex- amples of suitable carriers are water, salt solutions, alcohols, polyethylene glycols, polyhy- droxyethoxylated castor oil, peanut oil, olive oil, gelatine, lactose, terra alba, sucrose, glu- cose, cyclodextrin, amylose, magnesium stearate, talc, gelatin, agar, pectin, acacia, stearic acid or lower alkyl ethers of cellulose, silicic acid, fatty acids, fatty acid amines, fatty acid monoglycerides and diglycerides, pentaerythritol fatty acid esters, polyoxyethylene, hy-

droxymethylcellulose and polyvinylpyrrolidone. Similarly, the carrier or diluent may include any sustained release material known in the art, such as glyceryl monostearate or glyceryl distearate, alone or mixed with a wax. The formulations may also include wetting agents, emulsifying and suspending agents, preserving agents, sweetening agents, thickeners or fla- vouring agents. The formulations of the invention may be formulated so as to provide quick, sustained, or delayed release of the active ingredient after administration to the patient by employing procedures well known in the art.

The pharmaceutical compositions can be sterilized and mixed, if desired, with auxiliary agents, emulsifiers, salt for influencing osmotic pressure, buffers and/or colouring sub- stances and the like, which do not deleteriously react with the active compounds.

The route of administration may be any route, which effectively transports the active com- pound to the appropriate or desired site of action, such as oral, nasal, rectal, pulmonary, transdermal or parenteral e. g. depot, subcutaneous, intravenous, intraurethral, intramuscular, intranasal, ophthalmic solution or an ointment, the parenteral or the oral route being pre- ferred.

If a solid carrier is used for oral administration, the preparation may be tabletted, placed in a hard gelatin capsule in powder or pellet form or it can be in the form of a troche or lozenge. If a liquid carrier is used, the preparation may be in the form of a suspension or solution in wa- ter or a non-aqueous media, a syrup, emulsion or soft gelatin capsules. Thickeners, flavor- ings, diluents, emulsifiers, dispersing aids or binders may be added.

For nasal administration, the preparation may contain a compound of formula I dissolved or suspended in a liquid carrier, in particular an aqueous carrier, for aerosol application. The carrier may contain additives such as solubilizing agents, e. g. propylene glycol, surfactants, absorption enhancers such as lecithin (phosphatidylcholine) or cyclodextrin, or preservatives such as parabenes.

For parenteral application, particularly suitable are injectable solutions or suspensions, pref- erably aqueous solutions with the active compound dissolved in polyhydroxylated castor oil.

Tablets, dragees, or capsules having talc and/or a carbohydrate carrier or binder or the like are particularly suitable for oral application. Preferable carriers for tablets, dragees, or cap-

sules include lactose, corn starch, and/or potato starch. A syrup or elixir can be used in cases where a sweetened vehicle can be employed.

In formulations for treatment or prevention of infectious diseases in mammals the amount of active modified PNA molecules used is determined in accordance with the specific active drug, organism to be treated and carrier of the organism.

Such mammals include also animals, both domestic animals, e. g. household pets, and non- domestic animals such as wildlife.

Usually, dosage forms suitable for oral, nasal, pulmonal or transdermal administration comprise from about 0.01 mg to about 500 mg, preferably from about 0.01 mg to about 100 mg of the compounds of formula I admixed with a pharmaceutical acceptable carrier or diluent.

In a still further aspect, the present invention relates to the use of one or more compounds of the general formula I or pharmaceutically acceptable salts thereof for the preparation of a medica- ment for the treatment and/or prevention of infectious diseases.

In yet another aspect of the present invention, the present invention concerns a method of treating or preventing infectious diseases, which treatment comprises administering to a pa- tient in need of treatment or for prophylactic purposes an effective amount of modified PNA according to the invention. Such a treatment may be in the form of administering a composi- tion in accordance with the present invention. In particular, the treatment may be a combina- tion of traditional antibiotic treatment and treatment with one or more modified PNA mole- cules targeting genes responsible for resistance to antibiotics.

In yet a further aspect of the present invention, the present invention concerns the use of the modified PNA molecules in disinfecting objects other than living beings, such as surgery tools, hospital inventory, dental tools, slaughterhouse inventory and tool, dairy inventory and tools, barbers and beauticians tools and the like.

EXAMPLES The following examples are merely illustrative of the present invention and should not be considered limiting of the scope of the invention in any way.

The following abbreviations related to reagents are used in the experimental part: (The monomers and the PNA sequences are stated in bold)

A monomer N- (2-Boc-aminoethyl)-N- (N6- (benzyloxycarbonyl) adenine-9- yl-acetyl) glycine Boc Tert butyloxycarbonyl Boc-Lys(2-CI-Z)-OH N-a-Boc-N-E-2-chlorobenzyloxycarbonyl-L-lysine C monomer N-(2-Boc-aminoethyl)-N-(N4-(benzyloxycarbonyl) cytosine-1- yl-acetyl)glycine TDBTU 2- (3, 4-dihydro-4-oxo-1,2,3-benzotriazin-3-yl)-1,1,3,3- tetramethyluronium tetrafluoroborate DICDiisopropylcarbodiimide HOBtN-Hydroxybenzotriazole DCCDicyclohexylcarbodiimide DCMDichloromethane DIEA N, N-diisopropylethylamine DMF N, N-dimethylformamide DMSO Dimethyl sulfoxide G monomer N-(2-Boc-aminoethyl)-N-(N2-(benzyloxycarbonyl) guanine-9- yl-acetyl)glycine HATUN- [ (1-H-benzotriazole-1-yl) (dimethylamine) methylene]-N- methylmethanaminiumhexafluorophosphate N-oxide HBTU 2- (1-H-benzotriazole-1-yl)-1, 1,3,3-tetramethyluronium hexafluorophosphate J monomer N- (2-Boc-aminoethyl)-N- (N-2- (benzyloxycarbonyl) isocyto- /nucleobasesine-5-yl-acetyl) glycine MBHA resin p-methylbenzhydrylamine resin NMP N-methyl pyrrolidone PTSA Para-Toluene sulphonic Acid Tmonomer N- (2-Boc-aminoethyl)-N- (thymine-1-yl-acetyl) glycine THF Tetrahydrofuran TFA Trifluoroacetic acid TFMSATrifluoromethanesulphonic acid Tris2-amino-2- (hydroxymethyl)-1, 3-propanediol Amino Acid Abbr. I Abbr. ll Sidechain Alanine A Ala methyl Arginine R Arg 3-guanidinopropyl Aspartic acid D Asp carboxymethyl Asparagine N Asn aminocarboxymethyl Cysteine C Cys mercaptomethyl Glutamic acid E Glu 2-carboxyethyl Glutamine Q Gln aminocarboxyethyl Histidine H His imidazol-4-yl-methyl Isoleucine I Ile 1-methylpropyl Leucine L Leu 2-methylpropyl Lysine K Lys 4-aminobutyl Methionine M Met 2- (methylthio) ethyl Phenylalanine F Phe benzyl Serine S Ser hydroxymethyl Threonine T Thr 1-hydroxyethyl Tryptophan W Try 3-indolyl Tyrosine Y Tyr 4-hydroxybenzyl Valine V Val 1-methylethyl Homoserine (Hse) 2-hydroxymethyl Citrulline (Cit) 3-ureidopropyl 4-pyridyl-alanine (4-Py) 4-pyridomethyl

The following abbreviations related to linking groups are used in the experimental part: (The linking groups as starting materials are indicated with capital letters whereas the linking groups in the finished peptide-PNA conjugate are indicated with small letters.) Abbreviation Linker (IUPAC) SMCCSuccinimidyl 4-(N-maleimidomethyl) cyciohexane-1-carboxylate LCSMCCSuccinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxy- (6- amido-caproate) MBS Succinimidyl m-maleimido-benzoylate EMCS Succinimidyl N-E-maleimido-caproylate SMPH Succinimidyl 6-(ß-maleimido-propionamido) hexanoate AMAS Succinimidyl N- (a-maleimido acetate) SMPBSuccinimidyl 4-(p-maleimidophenyl) butyrate P. ALA ! anine PHGPhenylglycine ACHC4-aminocyclohexanoic acid P. CYPR ß-(cyclopropyl) alanine AHA, AHEX 6-amino-hexanoic acid ADO, AEEA-OH ((2-aminoethoxy) ethoxy) acetic acid or 8-amino-3,6-dioxaoctanoic acid ADC Amino dodecanoic acid

The linking groups containing a succinimidyl group are shown in Figure 5.

All the linking groups are commercial available.

The composition of mixtures of solvents is indicates on a volume basis, i. e. 30/2/10 (v/v/v).

Preparative HPLC is performed on a DELTA PAK [Waters] (C18, 15 u. m, 300 A, 300x7.8 mm, 3 ml/min) A linear gradient from solvent A: 0.1 % TFA in water to B: 0.1 % TFA in acetonitrile was used. 0-2 min B 10 %, 2-30 min 40 % B, 30-35 min 100 % B, 35-37 min 100 % B, 37-38 min 10 % B, 37-50 min 10 % B.

Mass Spectrometry was performed on MALDI (Matrix Assisted Laser Desorption and lonisa- tion Time of Flight Mass Spectrometry) as HP MALDI-TOF # G2025A calibrated with peptide nucleic acids of the following weights: Mw, = 1584.5 g/mol, Mw2 = 3179.0 g/mol and Mus = 4605.4 g/mol.

Example 1 Monomer synthesis The monomers 4a-j were prepared from the protected D-amino acids as shown in the Scheme (26).

Scheme: Synthesis of the monomers 4a-j. R is given in Table 1 (backbone modification). R' = benzyl in a, b, e, f, i-k. R'= allyl in c, d, g, h. Reagents: a) BocNHCH2CHO, NaBH3CN, AcOH, MeOH ; b) thymin-1-yl-acetic acid, DCC, DhbtOH, DMF; c) 3a-e, j: H2, 10% Pd/C, MeOH ; 3g, h: Pd (PPh3) 4, morpholine, THF.

Oliqomerization : The PNA oligomers (Table 1) were synthesized as described (27) except that the guidelines (Chapter 3) to avoid racemization during coupling of the functional PNAs were followed : In the case of entry 4,5,9,10,12 and 14 the modified residues were success- fully incorporated by coupling with DIC/HOBt. In one of these cases (entry 12) the % racemi- zation was determined by the GC-MS method. As expected a very low amount of racemiza- tion (2.1 %) was detected. In the case of entry 6,7,8 and 11 the modified residues were in- corporated by coupling with TDBTU/DIEA, because coupling with DIC/HOBt failed to produce PNA oligomers that could be purified by RP-HPLC. For yet unknown reasons the monomer 4k (The 2-Py backbone modification: see Experimental) did not couple.

Hybridization properties The modified PNA dodecamers were examined for their binding to complementary, antiparallel RNA and DNA dodecamers by thermal stability (Tm) measure- ments (Table 1). Each modified monomer was incorporated in three positions. Several con- clusions can be drawn: i) Simple attachment of basic amino acids (entry 2,3) to the N-terminus of a PNA increases the Tm against both DNA and RNA: Probably the protonated amino and guanidinium groups form contacts to the last phosphate group in the oligonucleotide strand. ii) positioning the three amino groups (entry 6) or guanidinium groups (entry 7) inside the PNA strand dimin- ishes this effect: This is expected since 3D structures of PNA/RNA and PNA/DNA shows a too long distance between the phosphate and the charged residues (the nucleo bases are in the inside of the helix and the sugar and PNA backbone are at the outside). iii) A previous report (26) that the T-Lys increases the Tm against DNA could not be confirmed. iv) T-Arg are a better binder than T-Lys especially against DNA. v) This is a charge effect since PNA 2068 binds with much lower affinity towards NA vi) T-Leu is clearly the analogue that gives the lowest affinity towards DNA and RNA. Entry Backbone modifica-Coupling Selfmelt Tm Tm tion c reagent (Tx) (DNA) (RNA) 1 (PNA 2005) Gly HBTU 35.0 49.5 59.0 2 (PNA 2059) K3GIyd HBTU 46.0 52.0 62.5 3 (PNA 2058) R3GIye HBTU 44.5 52.0 62.5 4 (PNA 2062) Ala (5a) DIC/HOBt 34.0 48.5 58.0 5 (PNA 2060) Leu (5b) DIC/HOBt 30.0 42.5 52.5 6 (PNA 2066) Lys (5c) TDBTU 34.0 49.5 57.5 7 (PNA 2067) Arg (5d) TDBTU 40.0 51.5 58.5 8 (PNA 2068) Cit (5e) TDBTU 40.0 46.0 55.5 9 (PNA 2064) Gln (5f) DIC/HOBt 37.0 46.0 56.0 10 (PNA 2061) Ser (5g) DIC/HOBt nd nd 56. 0 11 (PNA 2069) Hse (5h) TDBTU nd 47. 5 57.0 12 (PNA 2063) Phe (5i) DlC/HOBt (0 37. 0 45.0 57.0 13 (PNA 2065) 4Py (5j) DIC/HOBt 34.0 45.5 55.5

Table 1. Melting temperatures (°C) of PNA-DNA and PNA-RNA duplexes. Measured in aqueous buffer containing 100 mM NaCI, 10 mM phosphate, 0.1 mM EDTA, pH 7.0; heating rate: 1 K min'. UV absorbance measured at 260 nm. The PNA sequence was H- TXACTxCATxACTCT-LysNH2 The complementary DNA sequences were: 5'- d (AGAGTATGAGTA). The complementary RNA sequences were: 5'-AGAGUAUGAGUA. (c) The backbone at the Tx position was constructed with the monomer derived from the indi- cated amino acid (20). (d) The PNA sequence was H-KKK-TxACTxCATxACTCT-LysNH2. (e) The PNA sequence was H-RRR-TxACTxCATxACTCT-LysNH2. (f) % racemisation (GC): 2.1.

Anti beta-lactamase activity : The results (Figure 7-10) shows relative ß-lactamase activity in converting nitrocefin. The values are the mean of four replicates with standard deviations in- dicated. The growth curves at 37 °C (not shown) shows that only PNA 2058 was slightly in- hibitory (1 fiM) to growth and only in the K12 strain (meaning that the PNAs were not toxic at the concentration used). The negative results regarding the K12 strain (Figure 7,8) prompted us to investigated the anti ß-lactamase activity in the more permeable AS19 strain (Figure 9,

10). In this case, both at 500 nM and at 1 tM concentration weak effects were in fact seen.

The more lipophilic (2060 and 2063) and the more basic (2058 and 2067) PNAs showed im- proved anti p-iactamase activity as compared to the parent PNA (2005). Since the more lipo- philic PNAs have low affinity towards RNA they probably excerted their effect by improved passive diffusion into the cells.

The use of cell-penetrating peptides to transport large molecules (including PNA) into eu- caryotic cells, has recently been reviewed (28). Similarly, it could be possible to improve up- take of PNAs in bacterial cells by conjugating the PNA to peptides.

Example 2 General : Boc-D-Hse (OBzl)-OH was from Advanced Chemtech. Boc-D-3- (2-pyridyl)-Ala-OH and Boc-D-3- (4-pyridyl)-Ala-OH were from Synthetech. Boc-D-Phe-OH, Boc-D-Ser (OBzl)- OH, Boc-D-Cit-OH and Boc-D-Arg (di-Z)-OH were from Bachem. Boc-D-Gln-OH, H-D-Leu- OBzl. PTSA, H-D-Ala-OBzl. PTSA were from NovaBiochem.

Antisense experiment: In vivo gene expression and inhibition by PNA was done by Dr. Liam Good (Stockholm) as described (24). The antisense data is for E. Coli strain K12 and AS19 grown in 100 L of Muller-Hinton broth.

Monomer synthesis : Alanine monomer (4a). 1a (4.00 g, 11.38 mmol) was freed from pTSA by addition of sat.

NaHCO3 (50 ml) and extraction with AcOEt (2 x 200 ml). The combined organic phases were washed with brine (25 ml) and dried (Na2SO4), and evaporated in vacuo to give 1.92 g (94%) of 1a. A solution of 1a (1.92 g, 10.71 mmol) and Boc-aminoacetaldehyde (1.70 g, 10.71 mmol) in MeOH (71 ml) were stirred at 0 °C for 5 min. NaCNBH3 (404 mg, 6.43 mmol) fol- lowed by AcOH (0.74 ml, 12.85 mmol) were added and the solution was stirred for 1 hr at rt.

The solution was evaporated in vacuo, sat NaHCO3 (75 ml) was added, and the mixture was extracted with AcOEt (250 ml). The organic phase was washed with brine (75 ml), and dried over Na2SO4, and the solvent evaporated in vacuo. 2a was purified by flash chromatography (AcOEt/hexane 2: 1). Yield: 2.50 g (72%) of 2a as an oil. 1 H-NMR (DMSO-d6) : 8 7.40 (m, 5H), 6.74 (t, 1H), 5.15 (s, 2H); 3.36 (m, 1 H), 2.96 (m, 2H), 2.53 (m, 2H), 2.12 (br. s, 1H), 1.39 (m, 9H), 1.20 (d, 3H).

DCC (2.55 g, 12.37 mmol) was added to a stirred solution of thymin-1-yl-acetic acid (2.28 g, 12.37 mmol) and DhbtOH (2.02 g, 12.37 mmol) in dry DMF (27 ml) at rt, and the solution was

stirred for 20 min at rt. 2a (2.49 g, 7.73 mmol) dissolved in dry DMF (27 ml) was added and the solution was stirred overnight. DCU was removed by filtration, and the DMF was re- moved in vacuo. The remaining oil was dissolved in AcOEt (200 ml), and the organic phase was washed with sat NaHCO3 (100 ml) and washed with brine (2 x 100 ml). The organic phase was dried (Na2SO4), and evaporated in vacuo to give a foam which was purified by flash chromatography (gradient of AcOEt (75-100%) in hexan). Yield: 3.59 g (95%) of 3a as a white foam.

3a (3.57 g, 7.31 mmole) in MeOH (94 ml) was hydrogenated at 1 atm. over 10% Pd-C (1160 mg) overnight. The solution was filtered through celite, and then evaporated in vacuo. Yield: 2.70 g (93%). Total yield from 1a : 42%. [a] D24= 18. 1 (c = 3 in MeOH).'H-NMR (DMSO-d6, major rotamer only) : 8 12.65 (br. s, 1 H), 11. 29 (s, 1 H), 7.35 (s, 1 H), 6.94 (m, 1 H), 4.62 (s, 2H), 4.33 (q, 1 H), 3.45-2.80 (m, 4H), 1.75 (s, 3H), 1.40 (m, 3H), 1.38 (s, 9H). FAB HRMS: m/z calcd. for C17H27N407 : 399.1880; found: 399.1884.

Leucine monomer (4b). 1 b (3.00 g, 7.62 mmole) was freed from pTSA by addition of sat NaHCO3 (100 ml) and extraction with DCM (3 x 200 ml). The combined organic phases were dried (Na2SO4), and evaporated in vacuo to give 1.69 g (100%) of 1b. A solution of 1b (1.69 g, 7.62 mmol) and Boc-aminoacetaldehyde (1.22 g, 7.62 mmol) in MeOH (51 ml) were stirred at 0 °C for 5 min. NaCNBH3 (288 mg, 4.58 mmol) followed by AcOH (0.52 ml, 9.16 mmol) were added and the solution was stirred for 1 hr at rt. The solution was evaporated in vacuo, sat NaHCO3 (50 ml) was added, and the mixture extracted with AcOEt (200 ml). The organic phase was washed with brine (50 ml), and dried over Na2SO4, and the solvent evaporated in vacuo. 2b was purified by flash chromatography (AcOEt/hexane 1: 2). Yield : 2.44 g of 2b as an oil (88%).'H-NMR (DMSO-d6,) : 8 7.38 (m, 5H), 6.71 (t, 1H), 5.14 (s, 2H); 3.25 (t, 1H), 2.99 (m, 2H), 2.65-2.30 (m, 2H), 2.03 (br. s, 1H), 1.67 (m, 1H), 1.38 (m, 9H), 0.85 (t, 6H).

DCC (1.80 g, 8.75 mmol) was added to a stirred solution of thymin-1-yl-acetic acid (1.61 g, 8.75 mmol) and DhbtOH (1.43 g, 8.75 mmol) in dry DMF (23 ml) at rt, and the solution was stirred for 45 min at rt. 2b (2.40 g, 6.58 mmol) dissolved in dry DMF (23 ml) was added and the solution was stirred overnight. DCU was removed by filtration, and the DMF was re- moved in vacuo. The remaining oil was dissolved in AcOEt (200 ml), and the organic phase was washed with sat NaHCO3 (50 ml) and washed with brine (2 x 50 ml). The organic phase was dried (Na2SO4), and evaporated in vacuo to give a foam which was purified by flash chromatography (eluent : AcOEt/hexan 2: 1). Yield: 2.99 g of 3b as a white foam (86%). 3b (2.99 g) in MeOH (40 ml) was hydrogenated at 1 atm over 10 % Pd-C (480 mg) for 4 hr at rt.

The solution was filtered through celite, and then evaporated in vacuo. Yield: 2.48 g (100%).

Total yield from 1b : 76%. [a] D24= 21.2 (c = 3 in MeOH).'H-NMR (DMSO-d6, major rotamer

only) : 8 12. 75 (br. s, 1H), 11.31 (s, 1 H), 7.41 (s, 1H), 6.92 (s, 1H), 4.7-4.3 (m, 3H), 3.5-2.9 (m, 4H), 1.8-1.4 (m, 3H) 1.76 (s, 3H), 1.38 (s, 9H), 0.88 (m, 6H). FAB HRMS: m/z calcd. for C2oH33N407 : 441.2349; found: 441. 2358.

Lysine (2-CI-Z) monomer (4c). Cs2CO3 (3.30 g, 10.12 mmol) was added to a stirred solution of Boc-D-Lys (2-CI-Z)-OH (4.00 g, 9.64 mmol) in DMF (35 ml). After 15 min allyle bromide (1.53 g, 12.65 mmol) was added and the mixture was stirred overnight and then filtered through celite. The filtrate was evaporated in vacuo, and the residue dried in vacuo. Sat Na- HC03 (100 ml) was added, and the mixture was extracted with AcOEt (200 ml). The organic phase was washed with brine (2 x 100 ml), dried over Na2SO4, and evaporated in vacuo to give 4.40 g (100%) of Boc-Lys (2-CI-Z)-O-Allyle. TFA (7.4 ml) was added at 0 °C to a solution of Boc-Lys (2-CI-Z)-O-Allyle (4.39 g, 9.65 mmol) in CH2CI2 (7.4 ml). The solution was stirred for 60 min at rt. and then neutralized by sat NaHCO3 (150 ml). The mixture was extracted with CH2CI2 (200 ml), and AcOEt (200 ml), and the combined extracts were dried (Na2SO4), and the solvent was evaporated in vacuo to give 3.27 g (96%) of 1c as an oil.

A solution of 1c (3.20 g, 9.01 mmol) and Boc-aminoacetaldehyde (1.44 g, 9.01 mmol) in MeOH (60 ml) were stirred at 0 °C for 5 min. NaCNBH3 (340 mg, 5.41 mmol) followed by AcOH (0.62 ml, 10.81 mmol) were added and the solution was stirred for 1 hr at rt. The solu- tion was evaporated in vacuo, sat NaHCO3 (50 ml) was added, and the mixture extracted with AcOEt (200 ml). The organic phase was washed with brine (50 ml) and dried over Na2SO4, and the solvent evaporated in vacuo. 2c was purified by flash chromatography (AcOEt/hexane 3: 1). Yield : 3.39 g of 2c as an oil (76%).'H-NMR (CDCI3) : 8 7.39 (m, 2H), 7.28 (m, 2H), 5.89 (m, 1H), 5.31 (dd, 2H), 5.20 (s, 2H), 4.94 (br. s, 2H), 4.60 (dd, 2H), 3.21 (m, 5H), 2.76 (m, 1H), 2.56 (m, 1H), 1.80-1.40 (m, 7H), 1.43 (m, 9H).

DCC (2.22 g, 10.80 mmol) was added to a stirred solution of thymin-1-yl-acetic acid (1.99 g, 10.80 mmol) and DhbtOH (1.76 g, 10.80 mmol) in dry DMF (23 ml) at rt, and the solution was stirred for 45 min at rt. 2c (3.36 g, 6.75 mmol) dissolved in dry DMF (23 ml) was added and the solution was stirred overnight. DCU was removed by filtration, and the DMF was re- moved in vacuo. The remaining oil was dissolved in AcOEt (200 ml), and the organic phase was washed with half sat NaHCO3 (50 ml) and washed with brine (2 x 50 ml). The organic phase was dried (Na2SO4), and evaporated in vacuo to give 5.44 g which was purified by flash chromatography twice (eluent : AcOEt/hexan 5: 1, and 10% MeOH in DCM). Yield: 3.64 g of 3c as a white foam (77%). Pd (PPh3) 4 (59 mg, 0.051 mmol) was added to a solution of 3c (3.41 g, 5.13 mmol) and morpholine (4.44 ml, 50.9 mmol) in THF (51 ml). The solution was stirred 45 min at rt, and then evaporated in vacuo. The white foam (4.27 g) was dissolved in AcOEt (200 ml) and extracted with 10% citric acid (2 x 100 ml). The combined H20 phases

were extracted with AcOEt (200 ml). The combined organic phases (400 ml) were washed with brine (50 ml) and dried (Na2SO4). The solvent was removed to give a foam which was dissolved in AcOEt (30 ml) and then slowly added to hexan (300 ml) under stirring at O °C.

Yield: 3.06 g of 4c as a white solid (100%). Total yield from 1c 61%. [oc] D24= 19.1 (c = 3 in MeOH).'H-NMR (DMSO-d6, major rotamer only) : 8 12.60 (br. s, 1H), 11.30 (s, 1 H), 7.48-7.35 (m, 5H), 6.90 (m, 1 H), 5.09 (s, 2H), 4.65 (m, 2H), 4.25 (t, 1 H), 3.43-2.60 (m, 6H), 2.00-1.00 (m, 7H), 1.76 (s, 3H), 1.39 (s, 9H). FAB HRMS: m/z calcd. for C28H39CIN5Og : 624.2436; found: 624.2422.

Arginine (di-Z) monomer (4d). Cs2CO3 (3.15 g, 9.67 mmol) was added to a stirred solution of Boc-D-Arg (di-Z)-OH (5.00 g, 9.21 mmol) in DMF (33 ml). After 15 min allyle bromide (0.96 ml, 11.05 mmol) was added and the mixture was stirred for 4 hr and then filtered through celite. The filtrate was evaporated in vacuo, and the residue dried in vacuo. sat NaHCO3 (100 ml) was added, and the mixture was extracted with AcOEt (200 ml). The organic phase was washed with brine (2 x 100 ml), dried over Na2SO4, and evaporated in vacuo to give 5.27 g (98%) of Boc-Arg (di-Z)-O-Allyle. TFA (6.9 mi) was added at 0 °C to a solution of Boc-D- Arg (di-Z)-O-Allyle (5.24 g, 9.00 mmol) in CH2CI2 (6.9 ml). The solution was stirred for 60 min at rt. and then neutrelized by sat NaHCO3 (120 ml). The mixture was extracted with CH2CI2 (200 ml) and AcOEt (200 ml), and the combined extracts were dried (Na2SO4), and the sol- vent was evaporated in vacuo to give 4.29 g (99%) of 1d as an oil. A solution of 1d (4.26 g, 8.83 mmol) and Boc-aminoacetaldehyde (1.41 g, 8.83 mmol) in MeOH (60 ml) were stirred at 0 °C for 5 min. NaCNBH3 (333 mg, 5.30 mmol) followed by AcOH (0.61 ml, 10.6 mmol) were added and the solution was stirred for 1 hr at rt. The solution was evaporated in vacuo, sat NaHCO3 (50 ml) was added, and the mixture extracted with AcOEt (200 ml). The organic phase was washed with brine (50 ml) and dried over Na2SO4, and the solvent evaporated in vacuo. 2d was purified by flash chromatography (AcOEt/hexane 1: 1). Yield: 4.28 g of 2d as an oil (77%).'H-NMR (DMSO-d6) : 8 9.45 (br. s, 1H), 9.25 (br. s, 1H), 7.50-7.20 (m, 10H), 5.84 (m, 1H), 5.31-5.13 (m, 6H), 4.95 (br. s, 1H), 4.55 (d, 2H), 4.00 (t, 2H), 3.20 (t, 1H), 3.10 (m, 2H), 2.67 (m, 1 H), 2.45 (m, 1 H), 1.80-1.40 (m, 5H), 1.43 (m, 9H).

DCC (2.25 g, 10.92 mmol) was added to a stirred solution of thymin-1-yl-acetic acid (2.01 g, 10.92 mmol) and DhbtOH (1.78 g, 10.92 mmol) in dry DMF (23 ml) at rt, and the solution was stirred for 45 min at rt. 2d (4.27 g, 6.82 mmol) dissolved in dry DMF (23 ml) was added and the solution was stirred overnight. DCU was removed by filtration, and the DMF was re- moved in vacuo. The remaining oil was dissolved in AcOEt (200 ml), and the organic phase was washed with half sat NaHCO3 (50 ml) and washed with brine (2 x 50 ml). The organic phase was dried (Na2SO4), and evaporated in vacuo to give 6.46 g which was purified by

flash chromatography (eluent : AcOEt/hexan 2: 1). Yield : 3.42 g of 3d as a white foam (63%).

Pd (PPh3) 4 (49 mg, 0.0424 mmol) was added to a solution of 3d (3.36 g, 4.24 mmol) and morpholine (3.7 ml, 42.4 mmol) in THF (42 ml). The solution was stirred 45 min at rt, and then evaporated in vacuo. The white foam (4.21 g) was dissolved en AcOEt and extracted with 10% citric acid (2 x 100 ml), and washed with brine (100 ml) and dried (Na2SO4). The solvent was removed to give a foam which was dissolved in AcOEt (30 ml) and then slowly added to hexane (300 ml) under stirring at 0'C. Yield : 2.20 g of 4d as a white solid (72%).

Total yield from 1d : 35%. [a] D24= 15. 8 (c = 3 in MeOH).'H-NMR (DMSO-d6, major rotamer only) : 8 12.65 (br. s, 1H), 11.29 (s, 1H), 9.17 (broad s, 2H), 7.60-7.20 (m, 11 H), 6.84 (m, 1 H), 5.26 and 5.06 (2 x s, 4H), 4.61-4.32 (m, 3H), 3.87 (m, 2H), 3.60-2.60 (m, 4H), 2.00-1.40 (m, 4H), 1.74 (s, 3H), 1.39 (s, 9H). FAB HRMS: m/z calcd. for C36H45N7O": 752.3255; found: 752.3274.

Citrulline monomer (4e). Cs2CO3 (2.49 g, 7.63 mmol) was added to a stirred solution of Boc-D-Cit-OH (2.0 g, 7.27 mmol) in DMF (26 ml). After 15 min benzyl bromide (1.04 ml, 8.72 mmol) was added and the mixture was stirred for 1.5 hr and then filtered through celite. The filtrate was evaporated in vacuo, and the residue dried in vacuo. Sat NaHCO3 (25 ml) was added, and the mixture was extracted with AcOEt (3 x 100 ml), dried over Na2SO4, and evaporated in vacuo to give only 1.85 g which was purified by flash chromatography (eluent : 10% MeOH in CH2CI2). Yield : 1.03 g of Boc-D-Cit-OBzl (39%). TFA (2.1 ml) was added at 0 °C to a solution of Boc-D-Cit-OBzl (1.01 g, 2.76 mmol) in CH2CI2 (2.1 ml). The solution was stirred for 60 min at rt. and then evaporated. Ether was added and a white creamy solid ap- peared. Most of the excess TFA was removed by decanting the ether of. The compound was used in the next step without further purification. A solution of le. TFA (2.70 mmol) and Boc- aminoacetaldehyde (0.43 g, 2.70 mmol) in MeOH (18 ml) were stirred at 0 °C for 5 min.

NaCNBH3 (280 mg, 4.2 mmol) was added and the solution was stirred for 1 hr at rt. The solu- tion was evaporated in vacuo. 2e was purified by flash chromatography (10% MeOH in DCM). Yield: 664 mg of 2d as an oil (60%).'H-NMR (DMSO-d6) : 5 7.39 (m, 5H), 6.75 (t, 1H), 5.91 (t, 1 H), 5.37 (s, 2H), 5.14 (s, 2H), 3.24 (m, 1H), 2.95 (m, 4H), 2.65-2.30 (m, 2H), 2.25 (br. s, 1 H), 1.49 (m, 4H), 1.38 (m, 9H).

DCC (0.418 g, 2.03 mmol) was added to a stirred solution of thymin-1-yl-acetic acid (0.374 g, 2.03 mmol) and DhbtOH (0.331 g, 2.03 mmol) in dry DMF (5 ml) at rt, and the solution was stirred for 20 min at rt. 2e (0.664 mg, 1.62 mmol) dissolved in dry DMF (4 ml) was added and the solution was stirred overnight. DCU was removed by filtration, and the DMF was re- moved in vacuo. The resulting oil was purified by flash chromatography twice (eluent : 10% MeOH in DCM and 12% MeOH in AcOEt). Yield: 0.777 g of 3e as a white foam (83%). 3e

(0.752 g) in MeOH (28 ml) was hydrogenated at 1 atm over 10 % Pd-C (173 mg) for 1 hr at rt and the solution was filtered through celite, and then evaporated in vacuo. Yield: 0.540 g of 4e as a white solid (85%). Total yield from 1e : 41%. [a] D24= 13. 4 (c = 3 in MeOH).'H-NMR (DMSO-d6, major rotamer only) : 8 12.75 (br. s, 1 H), 11.31 (s, 1 H), 7.39 (s, 1 H), 6.90 (br. s, 1 H), 5.96 (t, 1 H), 5.38 (s, 2H), 4.65 (s, 2H), 4.30 (m, 1 H), 3.42-2.40 (m, 4H), 1.92-1.85 (m, 4H), 1.76 (s, 3H), 1.39 (s, 9H). FAB HRMS: m/z calcd. for C2oH32N608Na : 507.2179; found: 507.2167.

Glutamin monomer (4f). Cs2CO3 (5.54 g, 17.05 mmol) was added to a stirred solution of Boc-D-Gln-OH (4.00 g, 16.2 mmol) in DMF (58 ml). After 20 min benzyl bromide (3.58 g, 20.9 mmol) was added and the mixture was stirred for 2 hr and then filtered through celite.

The filtrate was evaporated in vacuo, and the residue dried in vacuo. Sat NaHCO3 (75 ml) was added, and the mixture was extracted with AcOEt (150 ml). The organic phase was washed with brine (75 ml), dried over Na2SO4, and evaporated in vacuo to give 5.42 g of crude product. Recrystallisation from hexane/ethyl acetate (2: 1,240 ml) resulted in a white precipitate of Boc-D-Gln-OBzl, yield 4.64 g (85%). TFA (10.6 ml) was added at 0 °C to a solu- tion of Boc-D-Gln-OBzl (4.64 g, 13.8 mmol) in CH2CI2 (10.6 ml). The solution was stirred for 60 min at rt. and then neutrelized by sat NaHCO3 (175 ml). The mixture was extracted with CH2CI2 (6 x 200 ml), AcOEt (3 x 200 ml), and ether (3 x 200 ml) and the combined extracts were dried (Na2SO4), and the solvent was evaporated in vacuo to give 2.54 g (78%) of 1f as an oil. A solution of 1f (2.51 g, 10.62 mmol) and Boc-aminoacetaldehyde (1.69 g, 10.62 mmol) in MeOH (71 ml) were stirred at 0 °C for 5 min. NaCNBH3 (400 mg, 6.37 mmol) fol- lowed by AcOH (0.73 ml, 12.75 mmol) were added and the solution was stirred for 1 hr at rt.

The solution was evaporated in vacuo, sat NaHCO3 (50 ml) was added, and the mixture ex- tracted with AcOEt (2 x 200 ml). The combined organic phases were dried over Na2SO4, and the solvent evaporated in vacuo. 2f was purified by flash chromatography (10% MeOH in AcOEt). Yield: 2.34 g of 2f as an oil (58%).'H-NMR (DMSO-d6) : 8 7.39 (m, 5H), 7.27 (s, 1 H), 6.75 (br. s, 2H), 5.15 (s, 2H); 3.23 (s, 1H), 2.98 (m, 2H), 2.65-2.30 (m, 2H), 2.20-2.10 (m, 3H), 1.90-1.60 (m, 2H), 1.39 (m, 9H).

DCC (2.03 g, 9.85 mmol) was added to a stirred solution of thymin-1-yl-acetic acid (1.81 g, 9.85 mmol) and DhbtOH (1.61 g, 9.85 mmol) in dry DMF (21 ml) at rt, and the solution was stirred for 45 min at rt. 2f (2.34 g, 6.16 mmol) dissolved in dry DMF (21 ml) was added and the solution was stirred overnight. DCU was removed by filtration, and the DMF was re- moved in vacuo. The remaining oil was dissolved in AcOEt (200 mi), and the organic phase was washed with sat NaHCO3 (50 ml). The H20 phase was extracted with AcOEt (2 x 100 ml). The combined organic phases were dried (Na2SO4), and evaporated in vacuo to give

3.74 g which was purified by flash chromatography twice (eluent : 10% MeOH in AcOEt end 5% meOH in CH2CI2). Yield : 2.17 g of 3f as a white foam (65%). 3f (2.16 g) in MeOH (28 ml) was hydrogenated at 1 atm over 10 % Pd-C (340 mg) for 5 hr at rt. 3f precipitated out, on the Palladium. More MeOH (75 ml) was added and the solution was filtered through celite, and then evaporated in vacuo. Yield : 1.09 g of 4f as a white solid (61 %). Total yield from 1f 23%.

[a] D24 = 33.5 (c = 3 in DMF).'H-NMR (DMSO-d6, major rotamer only): 8 12.80 (br. s, 1 H), 11.32 (s, 1 H), 7.39-6.80 (m, 4H), 4.60 (m, 2H), 4.25 (m, 1 H), 3.45-2.45 (m, 4H), 2.20-1.95 (m, 4H), 1.78 (s, 3H), 1.40 (s, 9H). FAB HRMS: m/z calcd. for CigHsoNgOs : 456.2094; found: 456.2099.

Serine (OBzl) monomer (4g). Cs2CO3 (4.66 g, 14.3 mmol) was added to a stirred solution of Boc-D-Ser (OBzl)-OH (4.00 g, 13.6 mmol) in DMF (48 ml). After 15 min allyle bromide (2.20 g, 18.2 mmol) was added and the mixture was stirred for overnight and then filtered through celite. The filtrate was evaporated in vacuo, and the residue dried in vacuo. Sat. aq. NaHCO3 (100 ml) was added, and the mixture was extracted with AcOEt (200 ml). The organic phase was washed with brine (2 x 100 ml), dried over Na2SO4, and evaporated in vacuo to give 4.19 g of Boc-D-Ser (OBzl)-Oalylle (92%). TFA (9.6 ml) was added at 0 °C to a solution of Boc-D-Ser (OBzl)-Oallyle (4.19 g, 12.5 mmol) in CH2CI2 (9.6 ml). The solution was stirred for 60 min at rt. and then neutrelized by Sat. aq. NaHCO3 (170 ml). The mixture was extracted with CHzCIz (250 ml) and AcOEt (250 ml), and the combined extracts were dried (Na2SO4), and the solvent was evaporated in vacuo to give 2.99 g (100%) of 1g as an oil.

A solution of 1g (2.97 g, 12.64 mmol) and Boc-aminoacetaldehyde (2.01 g, 12.64 mmol) in MeOH (84 ml) were stirred at 0 °C for 5 min. NaCNBH3 (477 mg, 7.58 mmol) followed by AcOH (0.87 ml, 15.16 mmol) were added and the solution was stirred for 1 hr at rt. The solu- tion was evaporated in vacuo, sat NaHCO3 (50 ml) was added, and the mixture extracted with AcOEt (200 ml). The organic phases was washed with brine (50 ml) and dried over Na2SO4, and the solvent evaporated in vacuo. 2g was purified by flash chromatography (AcOEt/hexane 1: 1). Yield: 3.22 g of 2g as an oil (67%). Probably a weighing error. The yield is higher.

DCC (2.79 g, 13.54 mmol) was added to a stirred solution of thymin-1-yl-acetic acid (2.49 g, 13.54 mmol) and DhbtOH (2.21 g, 13.54 mmol) in dry DMF (29 ml) at rt, and the solution was stirred for 45 min at rt. 2g from above (3.20 g, 8.47 mmol. Probably more) dissolved in dry DMF (29 ml) was added and the solution was stirred overnight. DCU was removed by filtra- tion, and the DMF was removed in vacuo. The remaining oil was dissolved in AcOEt (200 ml), and the organic phase was washed with sat NaHCO3 (50 ml), and washed with brine (2 x 50 ml). The organic phase were dried (Na2SO4), and evaporated in vacuo to give 5.84 g

which was purified by flash chromatography twice (eluent : AcOEt: hexane 5: 1 and 10% MeOH in DCM). Yield : 4.60 g of 3g as a white foam (100%). Yield from 1g 67%. Pd (PPh3) 4 (115mg, 0.0995mm) was added to a solution of 3g (4. 60 g, 8.46 mmol) and morpholine (7.38 ml, 84.6 mmol) in THF (84 ml). The solution was stirred 2 hr at rt, and then evaporated in vacuo. The white foam was dissolved en AcOEt and extracted with 10% citric acid (2 x 100 ml), and washed withe brine (100 ml) and dried (Na2SO4). The solvent was removed to give a foam which was dissolved in AcOEt (30 ml) and then slowly added to hexan (300 ml) under stirring at O C. Yield: 3.79 g of 4g as a white solid (89%). Total yield from 1g : 60%. [a] D24 = 42.1 (c = 3 in MeOH).'H-NMR (DMSO-d6, major rotamer only) : Ob 12. 70 (br. s, 1 H), 11.31 (s, 1 H), 7.35 (m, 6 H), 6.85 (m, 1 H), 4.69-4.51 (m, 5H), 3.87 (m, 2H), 3.47-3.24 (m, 4H), 1.77 (s, 3H), 1.40 (s, 9H). FAB HRMS: m/z calcd. for C24H33N408 : 505.2298; found: 505.2310.

HomoSerine (OBzl) monomer (4h). Cs2CO3 (4.42 g, 13.57 mmol) was added to a stirred solution of Boc-D-Hse (OBzl)-OH (4.00 g, 12.92 mmol) in DMF (46 ml). After 15 min Allyle bromide (2.21 g, 18.2 mmol) was added and the mixture was stirred overnight and then fil- tered through celite. The filtrate was evaporated in vacuo, and the residue dried in vacuo. Sat NaHCO3 (100 mi) was added, and the mixture was extracted with AcOEt (200 ml). The or- ganic phase was washed with brine (2 x 100 ml), dried over Na2SO4, and evaporated in vacuo to give 4.44 g (98%) of product. TFA (9.6 ml) was added at 0 °C to a solution of Boc- D-Hse (OBzl)-Oallyle (4.34 g, 12.4 mmol) in CH2CI2 (9.6 ml). The solution was stirred for 60 min at rt. and then neutrelized by Sat. aq. NaHCO3 (170 ml). The mixture was extracted with CH2CI2 (250 ml) and AcOEt (250 ml), and the combined extracts were dried (Na2SO4), and the solvent was evaporated in vacuo to give 2.93 g (95%) of 1 h as an oil.

A solution of 1 h (2.92 g, 11.71 mmol) and Boc-aminoacetaldehyde (1.86 g, 11.71 mmol) in MeOH (78 ml) were stirred at 0 °C for 5 min. NaCNBH3 (442 mg, 7.03 mmol) followed by AcOH (0.80 ml, 14.05 mmol) were added and the solution was stirred for 1 hr at rt. The solu- tion was evaporated in vacuo, sat NaHCO3 (50 ml) was added, and the mixture extracted with AcOEt (200 ml). The organic phases was washed with brine (50 ml) and dried over Na2SO4, and the solvent evaporated in vacuo. 2g was purified by flash chromatography (AcOEt/hexane 1: 1). Yield : 3.28 g of 2h as an oil (71%).'H-NMR (CDC13) : 8 7.32 (m, 5H), 5.87 (m, 1 H), 5.33-5.20 (m, 2H), 4.99 (br. s, 1 H), 4.58 (d, 2H), 4.49 (s, 2H), 3.57 (m, 2H), 3.43 (t, 1H), 3.15 (m, 2H), 2.78 (m, 1H), 2.57 (m, 1H), 2.00 (m, 1H), 1.86 (m, 1H), 1.64 (br. s, 1 H), 1.44 (m, 9H).

DCC (2.70 g, 13.09 mmol) was added to a stirred solution of thymin-1-yl-acetic acid (2.41 g, 13.09 mmol) and DhbtOH (2.13 g, 13.09 mmol) in dry DMF (28 mi) at rt, and the solution was stirred for 45 min at rt. 2h (3.21 g, 8.18 mmol) dissolved in dry DMF (28 ml) was added and

the solution was stirred overnight. DCU was removed by filtration, and the DMF was re- moved in vacuo. The remaining oil was dissolved in AcOEt (200 ml), and the organic phase was washed with sat NaHCO3 (50 ml), and washed with brine (2 x 50 ml). The organic phase were dried (Na2SO4), and evaporated in vacuo to give 5.79 g which was purified by flash chromatography twice (eluent : AcOEt: hexane 4: 1 and 10% MeOH in DCM). Yield : 3.96 g of 3h as a white foam (87%). Pd (PPh3) 4 (82 mg, 0.0708 mmol) was added to a solution of 3h (3.96 g, 7.08 mmol) and morpholine (6.2 ml, 70.8 mmol) in THF (70 ml). The solution was stirred 30 min at rt, and then evaporated in vacuo. The white foam was dissolved in AcOEt (200 ml) and extracted with 10% citric acid (100 ml). The H20 phase was extracted with AcOEt (2 x 200 ml). The combined organic phases were washed with 10% citric acid (50 ml), brine (50 ml) and dried (Na2SO4). The solvent was removed to give a foam which was dis- solved in AcOEt (30 ml) and then slowly added to hexan (300 ml) under stirring at O °C.

Yield: 3.44 g of 4h as a white solid (94%). Total yield from 1 h : 58%. [a] D24 = 48.2 (c = 3 in MeOH).'H-NMR (DMSO-d6, major rotamer only) : Ob 12.65 (br. s, 1H), 11.31 (s, 1H), 7.34 (m, 6 H), 6.81 (m, 1 H), 4.63-4.46 (m, 4H), 4.20 (m, 1H), 3.60-3.22 (m, 6H), 2.10 (m, 1H), 2.05 (m, 1H), 1.76 (s, 3H), 1.40 (s, 9H). FAB HRMS : m/z calcd. for C25H35N408 : 519.2455; found: 519.2539.

Phenylalanine monomer (4i). Cs2CO3 (6.43 g, 19.7 mmol) was added to a stirred solution of Boc-D-Phe-OH (5.00 g, 18.8 mmol) in DMF (67 ml). After 20 min benzyl bromide (3.88 g, 22.6 mmol) was added and the mixture was stirred for 2 hr and then filtered through celite.

The filtrate was evaporated in vacuo, and the residue dried in vacuo. Sat NaHCO3 (100 ml) was added, and the mixture was extracted with AcOEt (200 ml). The organic phase was washed with brine (100 ml), dried over Na2SO4, and evaporated in vacuo. Pure Boc-D-Phe- OH was obtained by flash chromatography (Eluent : AcOEt: Hexane 1: 3). Yield 6.42 g, 96%.

TFA (14 ml) was added at 0 °C to a solution of Boc-D-Phe-OBzl (6.40 g, 18.0 mmol) in CH2CI2 (14 ml). The solution was stirred for 60 min at rt. and then neutrelized by Sat. aq.

NaHCO3 (200 ml). The mixture was extracted with CH2CI2 (2 x 200 ml) and AcOEt (2 x 200 ml), and the combined extracts were dried (Na2SO4), and the solvent was evaporated in vacuo to give 4.62 g (100%) of 1i as an oil. A solution of 1i (4.50 g, 17.62 mmol) and Boc- aminoacetaldehyde (2.81 g, 17.62 mmol) in MeOH (115 ml) were stirred at 0 °C for 5 min.

NaCNBH3 (0.664 g, 10.57 mmol) followed by AcOH (1.21 ml, 21.14 mmol) were added and the solution was stirred for 1 hr at rt. The solution was evaporated in vacuo, sat NaHCO3 (50 ml) was added, and the mixture extracted with AcOEt (200 ml). The organic phase was washed with brine (50 ml) dried over Na2SO4, and the solvent evaporated in vacuo. 2i was purified by flash chromatography (AcOEt: hexane 1: 1). Yield: 5.42 g of 2i as an oil (77%).'H-

NMR (CDC13) : 8 7.34-7.11 (m, 10H), 5.08 (s, 2H), 4.85 (br. s, 1H), 3.53 (t, 1H), 3.11 (t, 2H), 2.92 (m, 2H), 2.74 (m, 1 H), 2.60 (m, 1 H), 1.55 (br. s, 1 H), 1.43 (m, 9H).

DCC (4.44 g, 21.50 mmol) was added to a stirred solution of thymin-1-yl-acetic acid (3.96 g, 21.50 mmol) and DhbtOH (3.51 g, 21.50 mmol) in dry DMF (45 ml) at rt, and the solution was stirred for 45 min at rt. 2i (5.37 g, 13.44 mmol) dissolved in dry DMF (45 ml) was added and the solution was stirred overnight. DCU was removed by filtration, and the DMF was re- moved in vacuo. The remaining oil was dissolved in AcOEt (200 ml), and the organic phase was extracted with sat NaHCO3 (200 ml). The organic phase was washed with brine (200 ml), and dried (Na2SO4), and evaporated in vacuo to give 8.76 g foam which was purified by flash chromatography twice (eluent : AcOEt: hexane 2: 1). Yield: 6.88 g of 3i as a white foam (91%). 3i (6.78 g) in MeOH (86 ml) was hydrogenated at 1 atm over 10 % Pd-C (1.02 g) for 2 hr at rt. The solution was filtered through celite, and then evaporated in vacuo. Yield: 5.08 g of 4i as a white solid (89%). Total yield from 1i : 62%. [a] D24= 120. 7 (c = 3 in MeOH).'H-NMR (DMSO-d6, major rotamer only) : 8 12.65 (br. s, 1 H), 11.34 (s, 1 H), 7.38-7.24 (m, 6H), 6.60 (s, 1H), 4.56 (m, 2H), 4.16 (m, 1 H), 3.40-2.60 (m, 6H), 1.79 (s, 3H), 1.35 (s, 9H). FAB HRMS: m/z calcd. for C23H3, N407 : 475.2193; found: 475.2184.

4-pyridyl monomer (4j). Cs2CO3 (6.43 g, 19.7 mmol) was added to a stirred solution of Boc- D-4-Py-OH (5.00 g, 18.8 mmol) in DMF (67 ml). After 15 min benzyl bromide (3.75 g, 21.9 mmol) was added and the mixture was stirred overnight. The red reaction mixture was quenced by addition of half sat NaHCO3 (400 ml) and then extracted with AcOEt (3 x 200ml). The combined organic phases were washed with brine (2 x 100 ml), dried over MgS04, and evaporated in vacuo. Pure Boc-D-4-Py-OBzl was obtained by flash chromatog- raphy (Eluent : AcOEt: Hexane 2: 1). Yield 5.36 g (80%). TFA (11.6 ml) was added at 0 °C to a solution of Boc-D-4-Py-OBzl (5.35 g, 15.00 mmol) in CH2CI2 (11.6 ml). The solution was stirred for 60 min at rt. and then neutrelized by Sat. aq. NaHCO3 (200 ml). The mixture was extracted with CH2CI2 (2 x 200 ml) and AcOEt (2 x 200 ml), and the combined extracts were dried (MgS04), and the solvent was evaporated in vacuo to give 3.62 g (94%) of 1j as an oil.

A solution of 1j (3.45 g, 13.46 mmol) and Boc-aminoacetaldehyde (2.14 g, 13.46 mmol) in MeOH (90 ml) were stirred at 0 °C for 5 min. NaCNBH3 (0.507 g, 8.08 mmol) followed by AcOH (0.92 ml, 16.15 mmol) were added and the solution was stirred for 1 hr at rt. The solu- tion was evaporated in vacuo, sat NaHCO3 (50 ml) was added, and the mixture extracted with AcOEt (3 x 150 ml) and DCM (150 ml). The combined organic phases were dried over Na2SO4, and the solvent evaporated in vacuo. 2j was purified by flash chromatography (elu- ent: 5% MeOH in DCM). Yield: 4.27 g of 2j as an oil (80%).'H-NMR (CDC13) : 6 8.41 (d, 2H),

7.31-7.19 (m, 5H), 7.00 (d, 2H), 5.07 (s, 2H), 4.88 (br. s, 1H), 3.51 (t, 1H), 3.09 (m, 2H), 2.86 (d, 2H), 2.72 (m, 1H), 2.52 (m, 1H), 1.56 (br. s, 1 H), 1.41 (m, 9H).

DCC (3.43 g, 16.64 mmol) was added to a stirred solution of thymin-1-yl-acetic acid (3.07 g, 16.64 mmol) and DhbtOH (2.71 g, 16.64 mmol) in dry DMF (35 ml) at rt, and the solution was stirred for 45 min at rt. 2j (4.15 g, 10.40 mmol) dissolved in dry DMF (35 ml) was added and the solution was stirred overnight. DCU was removed by filtration, and the DMF was re- moved in vacuo. The remaining oil was dissolved in AcOEt (200 ml), and the organic phase was extracted with half sat NaHCO3 (50 ml). The H20 phase was extracted with AcOEt (2 x 50 ml). The combined organic phases were washed with brine (100 ml), and dried (Na2SO4), and evaporated in vacuo to give 7.19 g foam which was purified by flash chromatography twice (eluent : 10% MeOH in AcOEt, and 10% MeOH in DCM). Yield : 4.07 g of 3j as a white foam (69%). 3j (3.93 g, 6.96 mmol) in MeOH (50 ml) was hydrogenated at 1 atm over 10 % Pd-C (0.83 g) 4 hr at rt. 3j precipitated out, on the Palladium. More MeOH (400 ml) was added and the solution was filtered through celite, and then evaporated in vacuo. Yield: 2.80 g of 4j as a white solid (85%). Total yield from 1j : 47%. [a] 24 = 87.1 (c = 3 in MeOH).'H- NMR (DMSO-d6, major rotamer only) : 8 11.36 (s, 1 H), 8.47 (d, 2H), 7.39 (d, 2H), 6.73 (s, 1H), 4.56 (dd, 2H), 4.29 (m, 1 H), 3.40-2.60 (m, 6H), 1.78 (s, 3H), 1.36 (s, 9H). FAB HRMS: m/z calcd. for C22H30N507 : 476.2145; found: 476.2136.

2-pyridyl monomer (4k). Cs2CO3 (6.43 g, 19.7 mmol) was added to a stirred solution of Boc-D-2-Py-OH (5.00 g, 18.8 mmol) in DMF (67 ml). After 15 min benzyl bromide (4.06 g, 23.7 mmol) was added and the mixture was stirred overnight. The red reaction mixture was quenced by addition of half sat. NaHCO3 (400 ml) and then extracted with AcOEt (3 x 200ml). The combined organic phases were washed with brine (2 x 100 ml), dried over Na2SO4, and evaporated in vacuo. Pure Boc-D-2-Py-OBzl was obtained by flash chromatog- raphy (Eluent : AcOEt: Hexane 1: 1). Yield 6.54 g (98%). TFA (14 ml) was added at 0 °C to a solution of Boc-D-2-Py-OBzl (6.38 g, 17.9 mmol) in CH2CI2 (14 ml). The solution was stirred for 60 min at rt. and then neutrelized by Sat. aq. NaHCO3 (200 ml). The mixture was ex- tracted with CH2CI2 (2 x 200 ml) and AcOEt (2 x 200 ml), and the combined extracts were dried (MgS04), and the solvent was evaporated in vacuo to give 4.50 g (98%) of 1k as an oil.

A solution of 1k (4.50 g, 17.62 mmol) and Boc-aminoacetaldehyde (2.81 g, 17.62 mmol) in MeOH (115 ml) were stirred at 0 °C for 5 min. NaCNBH3 (0.664 g, 10.57 mmol) followed by AcOH (1.21 ml, 21.14 mmol) were added and the solution was stirred for 1 hr at rt. The solu- tion was evaporated in vacuo, sat NaHCO3 (100 ml) was added, and the mixture extracted with AcOEt (2 x 200 ml). The organic phase was dried over MgS04, and the solvent evapo-

rated in vacuo. 2k was purified by flash chromatography (eluent : A stepwise gradient of MeOH in AcOEt (0 to 5%)). Yield: 5.23 g of 2k as an oil (75%).'H-NMR (CDCI3) : 6 8.49 (d, 1H), 7.51 (m, 1H), 7.29 (m, 5H), 7.06 (m, 2H), 5.15 (br. s, 1H), 5.10 (s, 2H), 3.80 (dd, 1 H), 3.11 (m, 4H), 2.77 (m, 1H), 2.59 (m, 1H), 1.90 (br. s, 1H), 1.41 (m, 9H).

DCC (4.26 g, 20.65 mmol) was added to a stirred solution of thymin-1-yl-acetic acid (3.80 g, 20.65 mmol) and DhbtOH (3.37 g, 20.65 mmol) in dry DMF (43 ml) at rt, and the solution was stirred for 30 min at rt. 2k (5.15 g, 12.91 mmol) dissolved in dry DMF (43 ml) was added and the solution was stirred overnight. DCU was removed by filtration, and the DMF was re- moved in vacuo. The remaining oil was dissolved in AcOEt (200 ml), and the organic phase was extracted with half sat NaHCO3 (200 ml). The H20 phase was extracted with AcOEt (2 x 200 ml). The combined organic phases were washed with brine (200 ml), and dried (Na2SO4), and evaporated in vacuo to give 9.07 g foam which was purified by flash chroma- tography twice (eluent : A stepwise gradient of MeOH in AcOEt (0 to 5%), and 5% MeOH in DCM). Yield : 5.71 g of 3k as a white foam (78%).

3k (5.61 g, 9.90 mmol) in MeOH (71 mi) was hydrogenated at 1 atm over 10 % Pd-C (0.83 g) overnight at rt. The solution was filtered through celite, and then evaporated in vacuo. The resulting oil was dissolved in DCM (50 ml) and slowly added to hexan (500 ml) at 0 C. Yield : 4.18 g of 4k as a white solid (89%). Total yield from 1 k : 53%. [a] D24 = 86. 2 (c = 3 in MeOH).

'H-NMR (DMSO, major rotamer only) : 8 12.80 (br. s, 1 H), 11.33 (s, 1 H), 8.52 (d, 1 H), 7.71 (m, 1H), 7.36-7.18 (m, 3H), 6.80 (s, 1H), 4.57 (m, 3H), 3.47-2.80 (m, 6H), 1.78 (s, 3H), 1.38 (s, 9H). FAB HRMS: m/z calcd. for C22H3oN507 : 476.2145; found: 476.2136.

PNA synthesis : PNA oligomers were synthesized as described (27). PNA 2069 was only 66% pure on RP-HPLC. All other PNAs were pure. PNA 2005: [JBL fr 2+3]: MALDI-MS 3308 (calc. 3307). PNA 2059: [TT38b fr 4]: MALDI-MS 3689 (calc. 3690). PNA 2058: [TT38a fr 2]: MALDI-MS 3778 (calc. 3774). PNA 2062: [TT33 fr 7]: MALDI-MS 3349 (calc. 3348). PNA 2060: [TT34 fr 11] : MALDI-MS 3474 (calc. 3474). PNA 2066: [TT49 fr 4]: MALDI-MS 3521 (calc. 3519). PNA 2067 : [TT51 fr11] : MALDI-MS 3613 (calc. 3609). PNA 2068 : [TT53fr3] : MALDI-MS 3606 (calc. 3606). PNA 2064: [AP-769V fr 3]: MALDI-MS 3519 (calc. 3519). PNA 2061: [TT37 fr 5] :. MALDI-MS 3400 (calc. 3396). PNA 2069: [TT54 fr 4]: MALDI-MS 3438 (calc. 3438). PNA 2063: [AP789 fr 12]: MALDI-MS 3577 (calc. 3576). PNA 2065: [AP-76911 fr 2]: MALDI-MS 3576 (calc. 3576).

Example 3 Preparation of H-KFFKFFKFFK-ado-TTC AAA CAT AGT-NH2

The peptide-PNA-chimera H-KFFKFFKFFK-ado-TTC AAA CAT AGT-NHz was synthesized on 50 mg MBHA resin (loading 100 pmol/g) (novabiochem) in a 5 mi glass reactor with a D-2 glassfilter. Deprotection was done with 2x600 jlL TFA/m-cresol 95/5 followed by washing with DCM, DMF, 5% DIEA in DCM and DMF. The coupling mixture was 200 pl 0.26 M solu- tion of monomer (Boc-PNA-T-monomer, Boc-PNA-A-monomer, Boc-PNA-G-monomer, Boc- PNA-C-monomer, Boc-AEEA-OH (ado) (PE Biosystems Inc.)) in NMP mixed with 200 aI 0. 5 M DIEA in pyridine and activated for 1 min with 200 pl 0. 202 M HATU (PE-biosystems) in NMP. The coupling mixture for the peptide part was 200 jli 0. 52 M NMP solution of amino acid (Boc-Phe-OH and Boc-Lys (2-CI-Z)-OH (novabiochem)) mixed with 200 u. i 1 M DIEA in NMP and activated for 1 min with 200 jli 0. 45 M HBTU in NMP. After the coupling the resin was washed with DMF, DCM and capped with 2 x 500 ll NMP/pyridine/acetic anhydride 60/35/5. Washing with DCM, DMF and DCM terminated the synthesis cycle. The oligomer was deprotected and cleaved from the resin using"low-high"TFMSA. The resin was rotated for 1 h with 2 ml of TFA/dimethylsulfid/m-cresol/TFMSA 10/6/2/0.5. The solution was re- moved and the resin was washed with 1 ml of TFA and added 1.5 ml of TFMSA/TFA/m- cresol 2/8/1. The mixture was rotated for 1.5 h and the filtrated was precipitated in 8 ml di- ethylether.

The precipitate was washed with 8 ml of diethylether. The crude oligomer was dissolved in water and purified by HPLC. Preparative HPLC was performed on a DELTA PAK [Waters ] (Cl 8, 15 pm, 300 A, 300x7.8 mm, 3 ml/min) A linear gradient from solvent A : 0.1 % TFA in water to B: 0.1 % TFA in acetonitrile was used. 0-2 min B 10 %, 2-30 min 40 % B, 30-35 min 100 % B, 35-37 min 100 % B, 37-38 min 10 % B, 37-50 min 10 % B.

Mw calculated : 4791.9 g/mol ; found on MALDI : 4791 g/mol.

Example 4 Maleimide activation of PNA PNA-oligomer ado-TTC AAA CAT AGT-NH2 (purified by HPLC) (2 mg, 0.589 -mol, Mw 3396.8) was dissolved and stirred for 15 min in NMP: DMSO 8: 2 (2 ml). Succinimidyl 4- (N- maleimidomethyl) cyclohexane-1-carboxylate (SMCC) (PIERCE) (1.1 mg, 3.24 mol, 5.5 eq.) dissolved in NMP (50 aI) and DIEA (34.7 ul, 198.7 limol) was added to the solution : The re- action mixture was stirred for further 2.5 h. The product was precipitated in diethylether (10 mL). The precipitate was washed with ether: NMP; 10: 1 (3x10mL) and ether (3x10mL).

Mw calculated: 3615.8 g/mol ; found on MALDI : 3613.5 g/mol.

The product was used without further purification.

Example 5 Conjugation of peptide and maleimide activated PNA

A solution of peptide CKFFKFFKFFK (0.5 mg in 200 ul degassed Tris buffer 10mM, pH 7.6 (329 nM)) was added to a solution of the above activated product (0.2 mg in 200 J DMF: Water 1: 1). The reaction mixture was stirred over night. The target compound was puri- fied by HPLC directly from the crude reaction mixture. Preparative HPLC was performed on a DELTA PAK [Waters] (C18, 15 um, 300 A, 300x7.8 mm, 3 ml/min) A linear gradient from sol- vent A: 0.1 % TFA in water to B: 0.1 % TFA in acetonitrile was used. 0-2 min B 10 %, 2-30 min 40 % B, 30-35 min 100 % B, 35-37 min 100 % B, 37-38 min 10 % B, 37-50 min 10 % B.

Mw calculated : 5133.0 g/mol ; found on MALDI : 5133 g/mol.

Example 6 H-LLKKLAKALKG-ahex-ado-CCATCTAATCCT-NH2 Performed in accordance with example 3, however with the use of 6-aminohexanoic acid (ahex) as linker together with 8-amino-3,6-dioxaoctanoic acid (ado).

Example 7 Preparation of H-KFFKFFKFF-ado-JTJTJJT-ado-ado-ado-TCCCTCTC-Lys- NH2 Performed in accordance with example 3, however with the use of PNA oligomer ado- JTJTJJT-ado-ado-ado-TCCCTCTC-Lys-NH2 instead of ado-TTC AAA CAT AGT-NH2.

This PNA is a triplex forming bis-PNA in which C (cytosine) in the"Hoogsteen strand"is ex- changed with the J nucleobases (a substitute for protonated C). This substitution assures efficient triplex formation at physiological pH (Egholm, M.; Dueholm, K. L. ; Buchardt, O. ; Coull, J.; Nielsen, P. E.; Nucleic Acids Research 1995,23,217-222, (125).

Example 8 The following oligomers and conjugates are prepared as described above: H-KFFKFFKFFK-achc-b. ala-TTCAAACAT (lys) AGT-NH2 T (lys) T (lys) CTAACATTTA-NH2 T (phe) T (lys) CTAACATTTA-NH2 T (lys) T (phe) CTAACATTTA-NH2 T (phe) T (phe) CTAACATTTA-NH2 T (lys) T (lys) C (phe) TAACATTTA-NH2 T (phe) T (lys) C (phe) TAACATTTA-NH2 T (lys) T (phe) C (phe) TAACATTTA-NH2 TT (phe) T (phe) C (phe) TAACATTTA-NH2

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