Synthesis of PNA oligoether conjugates

Article abstract of DOI:10.3390/molecules19033135

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.

Full text of DOI:10.3390/molecules19033135

Molecules 2014, 19, 3135-3148; doi:10.3390/molecules19033135
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthesis of PNA Oligoether Conjugates
Alice Ghidini 1, Peter Steunenberg 2, Merita Murtola 1,3 and Roger Strömberg 1,*
1
Department of Biosciences and Nutrition, Karolinska Institutet, Novum, Hälsovägen Huddinge 7,
14183, Sweden; E-Mails: alice.ghidini@ki.se (A.G.); merita.murtola@ki.se (M.M.)
2
ICL-IP Terneuzen, Frankrijkweg BJ, Terneuzen 6 4538, The Netherlands;
E-Mail: SteunenbergP@icl-ip.com
3
Department of Chemistry, University of Turku, Turku 20014, Finland
* Author to whom correspondence should be addressed; E-Mail: roger.stromberg@ki.se;
Tel.: +46-8-5248-1024; Fax: +46-8-5248-1034.
Received: 21 January 2014; in revised form: 1 March 2014 / Accepted: 4 March 2014 /
Published: 13 March 2014
Abstract: 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.
Keywords: peptide nucleic acid; polyethyleneglycol; aminosugar; neocuproine
1. 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

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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) [18–20] as
well as PNA-based artificial nucleases (PNAzymes) [21,22].
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.
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 [18–22] is performed as well as conjugation of an aminosugar.

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2. 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.
Scheme 1. Synthesis of PNA 2 and PNA 3.
(i) 1% Trifluoroacetic acid (TFA) in DCM, 5 times for 1 min; (ii) CDI in N-Methyl-2-pyrrolidone (NMP),
NMM, 1 h; (iii) oligoether 1; (iv) TFA/Triisopropylsilane (TIS)/H2O (95/2.5/2.5) for 2 h; (v) CDI in NMP,
NMM, 1 h, 2 times.
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 Tm values for the

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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 (Tm = 54 °C) of the same sequence, the oligoethers seem to be
accommodated at a reasonable trade-off of Tm 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)/1-
hydroxybenzotriazole 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% NH3/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 Tm of 51 °C for the RNA complex with
PNA 5 (Tm 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.
Scheme 2. Synthesis of oligoether building block 4 (A), tetraoligoether PNA conjugate
PNA 5 (B), aminosugar 7 (C) and PNA-aminosugar conjugate PNA 7 (D).
(A)
(B)

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Scheme 2. Cont.
(C)
(D)
(A) (i) Triethylene glycol: Benzoyl chloride 3:1, pyridine; (ii) Acetone, H2SO4 (1.5 M), Cr2O3; (B) (iii) 20%
Piperidine in NMP; (iv) oligoether 4, HBTU, HOBt, NMM; (v) 1% TFA in DCM, 5 times for 1 min; (vi)
oligoether 4, HBTU, HOBt, NMM; (vii) 20% NH3/MeOH, 5 h, 50 °C; (viii) TFA/TIS/H2O (95/2.5/2.5), 2 h;
(C) (ix) protected glycolic acid 6, dry CH2Cl2, TMSOTf, from 20 °C to room temperature, 3 h;
(x) Pd-C catalyst in THF-MeOH (1:1), overnight; and (D) (xi) 1% TFA in DCM, 5 times for 1 min;
(xii) Aminosugar 5, HATU, DIPEA in NMP, 1h; (xiii) TFA/TIS/H2O (95/2.5/2.5) for 2 h; (xiv) Ammonia,
5 h, 50 °C.
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 [26–28]. 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 Tm of 51 °C for the RNA complex with PNA 7 (Tm 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

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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
Zn2+ [18–21] or Cu2+ [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 Tm of 52 °C
for the RNA complex with PNA 9 (Tm 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.
Scheme 3. Synthesis of amino acid 8 (A), neocuproine and oligoether conjugate PNA 9
and PNA 10 (B).
(A)
O
O
O
(i, ii)
O
O
HO
O
O
O
O
HN
OH
HN
O
OH
TODA
HN
O
NH2
Fmoc-L-Dab-OH
O
O
O
8
(A) (i) Preactivation of TODA with HATU, NMM in DMF (2 min); (ii) Fmoc-L-Dab-OH; (B) Synthesis of
PNA 9 and PNA 10: (iii) 20% Piperidine in NMP, 30 min; (iv) acetic anhydride: lutidine: NMP 5:6:89
2 times for 5 min; (v) 1% TFA in DCM, 5 times for 1 min; (vi) 5-PhOC(O)NH-Neocuproine, NMM in NMP
overnight reaction; (vii) TFA/TIS/H2O (95/2.5/2.5) for 2 h; (viii) PEG 9, NMM, NMP.
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

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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 non-
conjugated 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.
Table 1. RP-18 retention times for PNA conjugates.
RP-18 HPLC
Ret. Time (min) a
Entry
PNA Construct
Type and Number of Conjugated Entities
1
2
3
4
5
6
7
8
9
PNA-Dapa-PNA 1
PNA 2
13.7
15.7
21.7
21.1
17.7
17.6
15.8
22.1
25.5
1 Dapa
1 hydroxyoligoether
PNA 3
2 hydroxyoligoethers
PNA 5
1 central and 3 terminal hydroxyoligoethers
1 Dapa
PNA-Dapa-PNA 6
PNA-Gly-PNA
PNA 7
1 glycine
1 aminosugar
PNA-GlyNeo-PNA
PNA 9
1 glycine and 1 neocuproine
1 methoxyoligoether and 1 neocuproine
1 methoxyoligoether and 1 neocuproine and
4 terminal methoxyoligoethers
10
PNA 10
27.9 b
a
Linear gradient of 0%–20% acetonitrile for 30 min. (with 0.1% TFA) on an Ascentis Express Supelco
Peptide ES-C18 (2,7 µm 150 × 4.6 mm) column at 60 °C with a flow rate of 1 mL/min; b A higher percentage
of acetonitrile was needed for this conjugate: Linear gradient of 7.5%–35% acetonitrile for
36 min. (with 0.1% TFA) on an Ascentis Express Supelco Peptide ES-C18 (2,7 µm 150 × 4.6 mm) column at
60 °C with a flow rate of 1 mL/min.
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).
3. Experimental
3.1. 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

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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.
3.2. Synthesis of PNAs
PNA 1, PNA 4, PNA 6, PNA 8, PNA-Gly-PNA and PNA-GlyDapa-PNA sequences
were assembled automatically on a solid support (Rink Amide resin) using the manufacturer’s
protocol for the Applied Biosystems 433A peptide synthesizer with 9-fluorenylmethyloxycarbonyl
(Fmoc)-chemistry and HATU (1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium
3-oxid hexafluorophosphate, from Iris Biotech) as coupling agent. PNA building blocks were from
Link Technologies Ltd (Strathclyde, UK) and Fmoc-αN-Lys (εN-Boc)OH was from Iris Biotech Gmbh
(Marktredwitz, Germany). PNAs were purified with an Ascentis Express Supelco Peptide ES-C18
(2, 7 µm 150 × 4.6mm) column at 60 °C using a flow rate of 1 mL/min and a linear gradient of 40% B
for 30 min. (A) 0.1% TFA–aq., (B) 0.1% TFA–aq., 50% MeCN.
PNA-Dapa-PNA 1 and PNA-Dapa-PNA 6 conjugates were obtained by cleaving PNA 1 and PNA
6, respectively, from support by TFA/TIS/water (95/2.5/2.5) (200 µL) for 2 h. Products were freeze
dried and purified on an Ascentis Express Supelco Peptide ES-C18 (2, 7 µm 150 × 4.6 mm) column at
60 °C using a flow rate of 1 mL/min and a linear gradient of 40% B for 30 min. (A) 0.1% TFA–aq.,
(B) 0.1% TFA–aq., 50% MeCN. (Retention time PNA-Dapa-PNA 1: 13.7 min and PNA-Dapa-PNA
6: 17.7 min). PNA-Dapa-PNA 1: C136H181N71O38 [M+], 3418; found, 3418 and PNA-Dapa-PNA
6: C129H166N68O37, 3259; found [M+] 3260.
PNA-Gly-PNA was obtained cleaving from support by TFA/TIS/water (95/2.5/2.5) (200 µL) for 2 h,
freeze dried and purified with a Ascentis Express Supelco Peptide ES-C18 (2, 7 µm 150 × 4.6 mm)
column at 60 °C using a flow rate of 1 mL/min and a linear gradient of 40% B for 30 min. (A) 0.1%
TFA–aq., (B) 0.1% TFA–aq., 50% MeCN. (Retention time PNA-Gly-PNA 1: 17.6 min). PNA-Gly-
PNA: C129H166N68O37, 3259; found [M+] 3260.
3.3. Post-Conjugation of Oligoethers to PNA
Synthesis of PNA 2 and PNA 3. Nβ-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)

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(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.
The PNA conjugates were cleaved from support by TFA/TIS/water (95/2.5/2.5) (200 µL) for 2 h,
freeze dried and purified with a Ascentis Express Supelco Peptide ES-C18 (2, 7 µm 150 × 4.6mm)
column at 60 °C using a flow rate of 1 mL/min and a linear gradient of 40% B for 30 min. (A) 0.1%
TFA–aq., (B) 0.1% TFA–aq., 50% MeCN. (Retention time PNA 2: 15.7 min and PNA 3: 21.7 min).
PNA 2: C143H194N72O42 [M+], 3591; found, 3590 and PNA 3: C150H207N73O46, 3767; found
[M+] 3769.
Synthesis of 2-(2-(2-hydroxyethoxy)ethoxy)ethyl benzoate (3). Triethylene glycol (3 mL, 3 eq, 30 mmol)
was twice dried by evaporation of added pyridine (20 mL). The residue was dissolved in pyridine (40 mL)
and benzoyl chloride (1.2 mL, 1 eq, 10 mmol) was then added dropwise to the dry solution at 0 °C and
at an interval of 10 min. After complete addition, the solution was allowed to obtain room temperature
and the reaction was monitored by TLC. After 2 h most of the reagent was converted and the reaction
was stopped by adding 5 mL of water and the pyridine was evaporated in the presence of added
acetonitrile. The crude material was dissolved in toluene and washed with NaHCO3 to remove excess
of the diol and the organic phase was dried over magnesium sulfate. The product was isolated by flash
chromatography on a column of silica gel using gradient hexane in ethyl acetate giving 1,02 g of 3
(40%). Mass calculated for compound 3, 254.12; found, [M+23(Na)] 277.46; 1H-NMR (CDCl3): δ
8.06 (d, 2H, J = 8); δ 7.56 (t, 1H, J = 7.4); δ 7.44 (t, 2H, J = 7.6); δ 4.50 (t, 2H, J = 4.7); δ 3.84 (t, 2H,
J = 5.1); δ 3.87 (m, 6H); δ 3.61 (t, 2H, J = 4.6).
Synthesis of 2-(2-(2-(benzoyloxy)ethoxy)ethoxy)acetic acid (4). Compound 3 (0.30 g, 1.2 mmol) was
dissolved in acetone (6 mL) and added dropwise to a solution of sulfuric acid (1.5 M, 7.2 mL)
containing chromium (VI) oxide (0.41 g, 4.1 mmol) at 0 °C. After complete addition of the alcohol, the
solution was kept at room temperature for 24 h. The chromium salts were removed by steps of
filtrations and centrifugations and the clear solution was concentrated under reduce pressure. The
crude material was extracted from the solution using toluene (3 × 40 mL) and dried over magnesium
sulfate. After filtration and concentration, the crude oil product was purified using column
chromatography on silica eluted with a gradient system running from pure dichloromethane with 1%
acetic acid to dichloromethane 90% methanol 10% with 1% acetic acid. This gave the product as
1
yellow oil (167 mg). Mass calculated for compound 4, 268.09; found, [M+23(Na)] 291.49; H-NMR
(CDCl3): δ 8.06 (d, 2H, J = 7.5); δ 7.56 (t, 1H, J = 7. 5); δ 7.44 (t, 2H, J = 7.5); δ 4.52 (t, 2H, J = 3.9);
δ 4.17 (s, 2H); δ 3.87 (t, 2H, J = 4.8); δ 3.76 (q, 2H, J = 5.4); 13C-NMR (CDCl3): δ 171.61.0; δ 165.55;
δ 132.10; δ 128.89; δ 128.76; δ 127.39; δ 76.32; δ 76.00; δ 75.69; δ 67.61; δ 62.69.
Synthesis of PNA 5. The terminal Fmoc was cleaved off by subjecting PNA 4 (4 mg, 1.13 µmol) to
20% piperidine in NMP for 25 min, followed by washing several times with DCM and NMP.

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Compound 4 (11.4 mg, 37.5 eq), HBTU (15.3 mg, 35.6 eq) and HOBt (5.4 mg, 35.6 eq) were
dissolved in 97.5 µL of NMP and NMM (8.25 µL, 65 eq) and agitated for 5 min to preactivate the
mixture, then added to the solid support and shaken for 30 min. The support was filtered and washed
with NMP and DCM. The Nβ-methyltrityl protection of the diaminopropionic acid was removed by
subjecting the solid support to 1% trifluoroacetic acid (TFA) in dichloromethane (DCM) for 5 × 1 min,
followed by washing with DCM and NMP. Compound 4 (34.2 mg, 112.5 eq), HBTU (45.9 mg, 106.8 eq)
and HOBt (16.2 mg, 106.8 eq) were dissolved in 318 µL of NMP and NMM (25.5 µL, 195 eq) were
added before agitation for 5 min. The preactivated mixture was added to the support and shaken for
30 min. The support was filtered and washed with NMP and DCM. The PNA conjugate was
deprotected using a 20% NH3/MeOH solution for 5 h at 50 °C and cleaved from support by
TFA/TIS/water (95/2.5/2.5) (200 µL) for 2 h, freeze dried and purified as for PNA 2 (Retention time
PNA 5: 21.1 min). Mass calculated for PNA 5: C164H231N75O55, 4131; found, [M+] 4131.
Synthesis of (7). To a solution of anhydrous CH2Cl2 (3 mL), 4 Å mol. sieves (0.1 g), protected glycolic
acid 6 (150 g, 0.91 mmol) and compound 5 (500 g, 0.91 mmol) was added, at 20 °C under argon
atmosphere, TMSOTf (15 µL, 0,075 mmol). Afterwards the reaction mixture was stirred at this
temperature for 3 h until TLC showed full conversion (PE:EtOAc 7:3, Rf 0.3). Than the mixture was
quenched by the addition of Et3N, filtered and the residue washed with CH2Cl2 (100 mL). The organic
layer was concentrated. Pd-C catalyst was added to a solution of the oily mixture obtained from the
previous reaction in 75 mL of (1:1) THF-MeOH. Afterwards, under stirring, the reaction mixture was
placed under an H2 gas atmosphere (atmospheric pressure) and the deprotection of the benzyl ester was
continued overnight. When full conversion had taken place (TLC examination), the H2 flow was
replaced by Argon and the reaction mixture was filtered and washed (MeOH, 200 mL) through a pad
of Celite. Upon concentration of the filtrate, a white solid material was obtained, and added CH2Cl2
was evaporated to remove traces of MeOH to afford 7 as a white solid (274 g, 0.6 mmol, 65% yield).
Mass calculated for compound 7, 459.10; found [M−] 458; 1H-NMR (DMSO-d6): δ 6.26 (1H, NH); δ
5.18 (1H, t, J = 10); δ 4.81 (1H, t, J = 10); δ 4.73 (1H, d, J = 8.4) ; δ 4.15–4.04 (2H, m); δ 3.93 (2H, s,
J = 2.4); δ 3.82–3.72 (2H, m); δ 1.98 (3H, s); δ 1.87 (3H, s); δ 1.83 (3H, s);. 13C-NMR (DMSO-d6): δ
171.1–169.3 (3 COCH3 and 1 COOH); δ 157.60; δ 114.2; δ 99.6; δ 77.0; δ 72.1; δ 70.6; δ 65.3; δ 62.0;
δ 54.0; δ 20.68; δ 20.65; δ 20.48.
Synthesis of PNA 7. The Nβ-methyltrityl protection of the diaminopropionic acid was removed
subjecting the solid support PNA 6 to 1% trifluoroacetic acid (TFA) in dichloromethane (DCM) for
5 × 1 min, followed by washing with DCM and NMP. Compound 7 (5.8 mg, 30 eq), HATU (4.3 mg,
27 eq) were dissolved in 100 µL of NMP and 2 µL of DIPEA and agitated for 1 min. The preactivated
mixture was added to the support and shaken for 1 h. The support was filtered and washed with NMP
and DCM. The PNA conjugate was cleaved from support by TFA/TIS/water (95/2.5/2.5) (200 µL) for
2 h and the carbohydrate protecting groups were removed using ammonia solution for 5 h at 50 °C.
The mixture was then lyophilized and purified as described for PNA 2 (Retention time PNA 7: 15.8 min).
Mass calculated for PNA 7: C138H183N71O42, 3507; found, [M+] 3508.

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3.4. Introduction of a New Amino Acid Building Block in the PNA Sequence
Synthesis of PNA-GlyNeo-PNA. Nβ-methyltrityl protection was cleaved off by subjecting the solid
supported PNA-GlyDapa-PNA to 1% trifluoroacetic acid in DCM for 5 × 1 min, followed by washing
with DCM and NMP. Then 5-phenoxycarbonylamino-2,9-dimethyl-1,10- phenanthroline (3.1 mg, 9 µmol),
NMM (5 µL, 50 µmol) in NMP (75 µL) was added to the support. The reaction was left overnight.
After washing with NMP and DCM, the PNA conjugate was cleaved from support by TFA/TIS/water
(95/2.5/2.5) (200 µL) for 2 h, freeze dried and purified (Retention time PNA-GlyNeo-PNA: 22.1 min).
Mass calculated for PNA-GlyNeo-PNA: C147H183N73O39, 3595; found, [M+] 3597.
Synthesis of (8). 2-(2-(2-methoxyethoxy)ethoxy]acetic acid (109 µL, 0.7 mmol, 1.2 eq) and HATU
(1.1 g, 1.9 eq) were dissolved in 2 mL of DMF and stirred for 2 min in presence of 0.5 eq of NMM and
then added to Fmoc-L-Dab-OH (200 mg, 0.58 mmol) previously suspended in 7 mL of DMF. The
reaction was run at room temperature for 20 min. The solution was dried and purified using column
chromatography on silica eluted with a gradient system running from pure dichloromethane with 1%
acetic acid to dichloromethane 90% methanol 15% with 0.1% acetic acid. This gave the product as 181
1
mg of a yellow oil. Mass calculated for compound 8 [M+], 500.54; found, [M+] 501.6; H-NMR
(CD3OD): δ 7.82 (d, 2H, J = 7.5); δ 7.71 (t, 2H, J = 6.6); δ 7.41 (t, 2H, J = 7.4); δ 7.34 (t, 2H, J = 7.4);
δ 4.40 (d, 2H, J = 6.3); δ 4.30–4.19 (m, 2H); δ 4.00 (s, 2H); δ 3.69-3.53 (m, 8H); δ 3.39 (s, 1H); δ 3.35
(s, 3H); δ 3.33 (s, 1H) δ 2.17–2.04 (m, 1H); δ 1.92–1.79 (m, 1H); 13C-NMR (CD3OD): δ 173.43; δ
159.10; δ 145.77; δ 129.20; δ 128.60; δ 126.70; δ 121.33; δ 73.26; δ 72.42; δ 71.70; δ 71.58; δ 69.81; δ
68.22; δ 59.46; δ 53.92; δ 48.77; δ 37.16; δ 32.73.
Synthesis of PNA 9. The terminal Fmoc was cleaved off by subjecting PNA 8 (5 mg, 1.04 µmol) to
20% piperidine in NMP for 25 min, followed by washing several times with DCM and NMP and the
terminal amino group was acetylated using a solution of acetic anhydride: lutidine: NMP, 5: 6: 89
(2 × 5 min). The support was filtered and washed with NMP and DCM. Nβ-methyltrityl protection was
cleaved off by subjecting the solid support to 1% trifluoroacetic acid in DCM for 5 × 1 min, followed
by washing with DCM and NMP then 5-phenoxycarbonylamino-2,9-dimethyl-1,10- phenanthroline
(3.1 mg, 9 µmol), NMM (5 µL, 50 µmol) and NMP (75 µL) were added to the support. The reaction
was left overnight. The PNA conjugate was cleaved from support by TFA/TIS/water (95/2.5/2.5) (200 µL)
for 2 h, freeze dried and purified (Retention time PNA 9: 25.5 min). Mass calculated for PNA 9:
C156H200N74O43, 3798; found, [M+] 3800.
Synthesis of PNA 10. The terminal Fmoc was cleaved off by subjecting PNA 8 (5 mg, 1.04 µmol) to
20% piperidine in NMP for 25 min, followed by washing several times with DCM and NMP and then
coupled with PEG 9 (8 mg, 5.8 µmol) in NMP (75 µL) with NMM (50 µmol, 5 µL) was added to the
support. The reaction was left overnight. The support was then filtered and washed with NMP and
DCM. Nβ-methyltrityl protection was cleaved off by subjecting the solid support to 1% trifluoroacetic
acid in DCM for 5 × 1 min, followed by washing with DCM and NMP then 5-phenoxycarbonylamino-
2,9-dimethyl-1,10- phenanthroline (3.1 mg, 9 µmol), NMM (50 µmol, 5 µL) and NMP (75 µL) were
added to the support. The reaction was left overnight.

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The PNA conjugate was cleaved from support by TFA/TIS/water (95/2.5/2.5) (200 µL) for 2 h,
freeze dried and purified with a Ascentis Express Supelco Peptide ES-C18 (2, 7 µm 150 × 4.6mm)
column at 60 °C using a flow rate of 1 mL/min and a linear gradient of 20%–100% B for 38 min.
(A) 0.1% TFA–aq., (B) 0.1% TFA–aq., 50% MeCN (Retention time PNA 10: 27.9 min). Mass
calculated for PNA 10: C210H301N81O66, 5013; found, [M+] 5014.
4. Conclusions
We have in this study demonstrated synthesis and approaches to PNA conjugated to different
oligoethers, an aminosugar and attachment of one or two different oligoether entities together with a
neocuproine derivative. The thermal melting is somewhat affected upon central positioning of the
oligoethers but this seems to be at an acceptable level for use of the conjugates. It is less clear how the
polarity of the conjugates is affected by addition of oligoethers (as judged from RP-HPLC retention)
and it is likely that how and where these are attached as well as the exact nature of the conjugated
entities play a role. Conjugation of oligoethers to PNA is in its infancy and is likely to influence
solubility and other properties of the PNA, perhaps also catalytic activity of PNAzymes and further
studies will reveal such influences.
Supplementary Materials
Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/19/3/3135/s1.
Acknowledgments
We greatly appreciate funding from the Swedish Science research council and the European 7th
framework program.
Conflicts of Interest
The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds PNA 2, PNA 3, PNA 5, PNA 7, PNA 9, PNA 10 and
compound 4, 7 and 8 are available from the authors.
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(http://creativecommons.org/licenses/by/3.0/).