Synthesis of PNA Oligoether Conjugates

Several different approaches have been explored for conjugation of oligoethers to PNA with internally or N-terminal placed diaminopropionic acid residues. Single and double conjugation of 2-(2-(2-aminoethoxy)ethoxy)ethanol was obtained using carbonyldimidazole. Using a post PNA-assembly coupling procedure the building block 2-(2-(2-(benzoyloxy)ethoxy)ethoxy)acetic acid multiple attachment of 2-(2-(2-hydroxyethoxy)ethoxy)acetyl groups to both N-terminal and β-amino groups of inserted diaminopropionic acids residues was achieved. Use of a new oligoether functionalized amino acid allows inclusion of oligoether conjugates during on-line machine assisted synthesis which also allowed combination of methods for attachment of different oligoethers and co-conjugation of neocuproine as well as conjugation of an aminosugar.


Introduction
Peptide nucleic acid (PNA) as first invented [1] are essentially N-(aminoethyl)glycine peptides carrying nucleic acid bases (aeg-PNA).PNA hybridizes to both DNA and RNA with Watson-Crick base pairing, thus mimicking the action of natural nucleic acids [2].A variety of modifications of the original backbone of PNA have been reported [3,4] and numerous studies directed at diagnostic use as well as consideration for therapeutics, has occurred over the past two decades [5,6].It is not uncommon for peptides, proteins, nanoparticles, oligonucleotides and even low molecular drugs to be conjugated to polyethyleneglykol (PEG) moieties in order to enhance uptake, solubility, stability, pharmacokinetics etc. for enhanced drug delivery [7,8].Conjugates to PNA typically involves conjugation to the carboxy or amino terminals either as exemplified for nanoparticle applications [9,10] or a nucleic acids delivery construct [11].Conjugation of shorter oligoethers to PNA is in general also to the carboxy or N terminal when the oligoether serves as a linker to other functionalities as in microRNA targeting PNA conjugated to a cellpenetrating peptide [12] or to the cleaver entity in a microRNA targeting PNA-based artificial nuclease [13].An interesting example of backbone inclusion of an oligoether into a peptide nucleic acid is γ-PNA, which is a modification that involves [2-(2-methoxyethoxy)ethoxy]methyl branching of the aminoethyl moiety of aeg-PNA.This backbone modification enhances affinity for the target nucleic acid as well the solubility of the PNA in aqueous media [14].
When a duplex between two oligonucleotides contains one or several unpaired nucleotides in an internal part of one of the strands a bulge is formed (Figure 1).The occurrence of one or several bulged out nucleotides in an otherwise complementary duplex usually results in substantial destabilization of the complex [15].Complexes with bulged out nucleotides can be stabilized by inclusion of modified nucleosides [16] or conjugation to entities that interact with the unpaired region [17].Bulged RNA is also known to be more susceptible to cleavage than RNA in a helical structure and has been interesting as targets for 2'-O-methyloligonucleotide artificial nucleases (OBAN's) [18][19][20] as well as PNA-based artificial nucleases (PNAzymes) [21,22].In the current study we present an investigation on the conjugation of oligoethers to PNA with internally or N-terminal placed diaminopropionic acid residues.We were concerned how the addition of oligoethers may affect the binding of a target RNA and other properties of the PNA.In particular, we were interested to see if different oligoether constructs would hamper formation of the complex between the PNA conjugate and RNA that forms a non-paired bulge region (Figure 1) and which constructs that can be accepted in future artificial nuclease designs.Therefore several different methods and constructs were developed as reported below.In addition co-conjugation of neocuproine that has been used in artificial nucleases [18][19][20][21][22] is performed as well as conjugation of an aminosugar.

Results and Discussion
The different approaches for the conjugation of oligoethers to PNA containing diaminopropionic acid (Dapa) were all performed with the PNA remaining on the solid support, where it was first assembled using Fmoc-chemistry.Prior to the post-conjugation, a 4-methyltrityl (Mtt) protecting group on the β-nitrogen of Dapa and/or the terminal Fmoc was removed.The methyltrityl (Mtt) group is acid-labile and readily removed by mild acidic treatment [23] and the amine is in situ neutralized in the subsequent conjugation reaction by adding an excess of base, such as N-Methylmorpholine (NMM) or N,N-Diisopropylethylamine.
First evaluated was a quick method that only involves commercial reagents, i.e., conjugation by reacting 1,1-carbonyldiimidazole (CDI, in a similar fashion as developed for oligonucleotide peptide conjugates [24]) with the Dapa β-amino group to produce intermediate PNA 1a and then add the amino-oligoether 1 to substitute the imidazole ring of the intermediate (Scheme 1).The reaction was not that effective but the product PNA 2 could be isolated by HPLC purification.Due to the incomplete conversion of PNA 1 we performed double treatment with CDI, reducing the amount of unreacted PNA but the amount of PNA 2 was not increased, instead a second product was formed that corresponds to the double substituted product PNA 3 (Scheme 1) which also could be isolated through HPLC purification.The CDI coupling procedure did produce both a mono and double conjugation product.However, the limited conversion, and somewhat demanding purification, made this procedure less recommendable.This suggested to us that we should look at other methodologies as well.Nevertheless PNA 2 and PNA 3 were isolated so that we could evaluate if the stability of the complex with RNA that forms a bulge upon hybridization to the PNA (Figure 1, right panel) is much affected.Thermal melting analysis with the target sequence UGUGUUCAUAAGCCC revealed that the T m values for the complexes was 49 °C for both PNA 2 and PNA 3.Although this is somewhat lower compared to the complex with the non-conjugated PNA (T m = 54 °C) of the same sequence, the oligoethers seem to be accommodated at a reasonable trade-off of T m and not interfering so severely that it would prevent association of the PNA with the RNA.
To get a method for postconjugation that was not only cleaner but also would allow multiple conjugations we then decided to utilize conditions similar to those for peptide synthesis with N,N,N′,N′-tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate (HBTU)/1hydroxybenzotriazole hydrate (HOBt) and an oligoether carrying a carboxylate function that can be coupled to the β-amino group of Dapa and/or the N-terminal amine.To obtain conjugates with a terminal hydroxyl group on the oligoether, compound 4 was synthesized by protecting one terminal of triethylene glycol with a benzoyl group and oxidizing the other hydroxyl group to a carboxylic acid (Scheme 2A).PNA 4 was synthesized and the Mtt groups removed with the PNA still on support whereupon coupling of oligoether 4 was performed by a procedure similar to that used for coupling of amino acids to the Dapa side chain [17,25], but with reduced excess of reagent (Scheme 2B).One could remove both Fmoc and Mtt protection before conjugation to obtain the fully oligoether conjugated PNA 5.However, to verify that orthogonal functionalization could be pursued we performed the side-chain and α-amino conjugation in separate steps.
In order to debenzoylate the oligoether the solid support was treated with 20% NH 3 /MeOH solution at room temperature overnight.However, after release of the PNA from the support the chromatogram and mass spectrum of the crude material revealed that debenzoylation was incomplete.Additional deprotection of the crude material with methanolic ammonia at 50 °C for 5 h gave complete conversion and PNA 5 was isolated by HPLC.Also for this PNA oligoether conjugate there was an even smaller effect of the conjugation of the four oligoether groups on the stability of the complex formed with the RNA sequence UGUGUUCAUAAGCCC, than found for the products obtained with the CDI reaction (see above).Thermal melting studies gave a T m of 51 °C for the RNA complex with PNA 5 (T m for the RNA complex with non-conjugated PNA = 54 °C) which suggests that there is less negative interference with the PNA/RNA complex than with the CDI linked hydroxyoligoether.Another class of compounds that can be interesting to conjugate to PNA, not only for increase of aqueous solubility, is sugars and aminosugars.As an example of this, and to evaluate if PNA/RNA complex with a bulge would be affected much by the presence of sugar, we performed a conjugation to a 2-aminoglucose derivative.Compound 5 was synthesized as described with initial protection of the amino group and successive acetylation of hydroxyl groups and adding a trichloroacetimidate group in the anomeric position [26][27][28].Subsequent glycosidation with a protected glycolic acid then gave product 7 [29,30].Reacting solid supported PNA 6 with an excess of compound 7 (30 eq) and HATU as condensing agent allowed us to obtain the aminosugar conjugate PNA 7. Thermal melting studies gave a T m of 51 °C for the RNA complex with PNA 7 (T m for the RNA complex with non-conjugated PNA = 54 °C).
In order to allow for more ready orthogonal conjugation of different groups we then turned to an alternative to post conjugation, where internal incorporation of an oligoether building block into PNA was evaluated.If this on-line approach is combined with the post-conjugations it would allow incorporation of three different conjugated entities, even though only Mtt and Fmoc temporary protection is used.To accomplish on-line incorporation we synthesized an oligoether carrying amino acid building block that can be inserted in the PNA sequence on the automated synthesizer.At the same time we also wished to see if a methoxy terminated oligoether would be accepted in PNA/RNA bulge complex.Thus, the commercially available oligoether 2-[2-(2-methoxyethoxy)ethoxy]-acetic acid (TODA) (Scheme 3A), which carries a terminal O-methyl group, was coupled with of HATU-preactivated Fmoc-L-Dab-OH (N-α-Fmoc-L-2,4-diaminobutyric acid) to obtain the oligoether amino acid building block 8.The amino acid 8 was then used in the synthesis of PNA 9 that is doubly conjugated with both an oligoether and neocuproine, which in the presence of a metal ion such as Zn 2+ [18][19][20][21] or Cu 2+ [22] turns into an RNA cleaver.The synthesis could be achieved by a standard PNA synthesis protocol on an automated synthesizer followed by removal of the Mtt from the Dapa unit and then post conjugation with 5-phenoxycarbonylaminoneocuproine [18,19] followed by removal of Fmoc, capping, cleavage from support and deprotection.Thermal melting studies gave a T m of 52 °C for the RNA complex with PNA 9 (T m for the RNA complex with non-conjugated PNA = 54 °C).Thus, an oligoether in this position can be acceptable in future artificial nuclease designs without substantial compromise with regard to the stability of the PNA/RNA bulge complex.We then wished to investigate if extensive multiple oligoether conjugation at the N-terminal would influence the stability of the complex and since the N-terminal Fmoc is orthogonal, additional complexity can be achieved by also conjugating at the amino terminal position.Thus, PNA 8 was converted to PNA 10 by reaction of the PNA with the reagent PEG 9 after removal of the Fmoc.PNA 10 was obtained but with this triply conjugated derivative carrying one neocuproine and five oligoether chains it was apparent that the solubility in water was problematic (to the extent that higher percentage of acetonitrile was necessary for the HPLC purification) and hence reliable thermal melting with the RNA complement could not be obtained.It appears that just addition of more oligoether chains does not necessarily give improved aqueous solubility.
In order to see if we could make sense of how the polarity was affected by oligoether conjugation we compared the RP-HPLC retention times for the conjugates and a couple of additional constructs (Table 1).PNA-Dapa-PNA 1 and PNA-Dapa-PNA 6 were obtained by complete deprotection of PNA 1 and PNA 6.These are as PNA 2 and PNA 3 or as PNA 7 respectively, but with a nonconjugated Dapa in the central position.In addition, PNA-Gly-PNA which is PNA-Dapa-PNA 6 with a glycine instead of the Dapa and PNA-GlyNeo-PNA which is as PNA 9 but with a glycine instead of the Dab-oligoether) without oligoethers were also synthesized.It is not an absolutely clear picture for these comparisons but conjugation of oligoethers seems to give a lower polarity, as judged by the shorter retention times during RP-HPLC analysis (cf.entries 1, 5, 6 and 8 with entries 2, 3, 4, 9 and 10).The only clear decrease of retention time, and presumably polarity, comes from conjugation of the aminoglucoside moiety (cf.entry 5-7).

Materials and Methods
Peptide nucleic acid monomers were from Link Technologies Ltd. (Bellshill, UK).Rink Amide resin was purchased from Biotage (Uppsala, Sweden).HBTU, HOBt and diaminopropionic acid and derivatives were purchased from Novabiochem (now incorporated into Merck Millipore, Darmstadt, Germany).Solvents and reagents for solid-phase synthesis were synthesis grade from Applied Biosystems (now incorporated into Life Technologies Europe, Stockholm, Sweden) and IRIS Biotech Gmbh (Marktredwitz, Germany).PEG 9 was purchased from Iris Biotech Gmbh.Other solvents were purchased from Merck Eurolab (Darmstadt, Germany).High-resolution mass spectrometry (HRMS) was performed on a Micromass LCT electrospray time-of-flight (ES-TOF) mass spectrometer in acetonitrile-water 1:1 (v/v) solutions.The molecular weights of the oligoribonucleotide and peptide nucleic acid conjugates were reconstructed from the m/z values using the mass deconvolution program of the instrument (Mass Lynx software package).The RNA substrate was purchased from Thermoscientific and was first purified by semi-preparative IE-HPLC (ion exchange high performance liquid chromatography) and then purified with RP-HPLC.Thermal melting analysis was determined from an absorbance vs. temperature profile measured at 260 nm on a Varian Cary 300 UV-vis dual beam spectrophotometer (Varian).Concentrations of both RNA and PNA were determined by UV absorption at 260 nm and calculated from extinction coefficients obtained by the nearest neighbor approximation [31].All chemicals used in the kinetics experiments were of molecular biology grade.

Post-Conjugation of Oligoethers to PNA
Synthesis of PNA 2 and PNA 3. N β -methyltrityl protection was cleaved off by subjecting the solid support bound PNA 1 (1. 4 µmol) to 1% trifluoroacetic acid (TFA) in dichloromethane (DCM) for 5 × 1 min, followed by washing with DCM and NMP.CDI (1, 1′-Carbonyldiimidazole) (0.014 mmol, 10 eq) was dissolved in NMP (150 µL) in a septum capped vial and NMM (4-Methylmorpholine) (0.071 mmol, 50 eq) was added to the solution.The mixture was added to the support bound PNA 1 through a septum and the reaction mixture was agitated for 1 h.The support was filtered but not washed.Polyether 1 (0.71 mmol) was then dissolved in NMP (150 µL) and added to the same support with 40 µL of NMM and shaken overnight.The support was filtered and washed with NMP and DCM.
PNA 3 was obtained when the CDI treatment was repeated twice and otherwise following the procedure for the oligoether 1 coupling as when isolating PNA 2.

Figure 1 .
Figure 1.Left panel: A tetranucleotide RNA-bulge (upper case letters) formed by incomplete base pairing upon binding to a partially complementary PNA-strand (lower case letters).Right panel: A tetranucleotide RNA-bulge (upper case letters) formed upon binding to a partially complementary PNA-strand with one or two amino acids (x) incorporated.