Non-nucleosidic analogues of polyaminonucleosides and their influence on thermodynamic properties of derived oligonucleotides

Article abstract of DOI:10.3390/molecules200712652

The rationale for the synthesis of cationic modified nucleosides is higher expected nuclease resistance and potentially better cellular uptake due to an overall reduced negative charge based on internal charge compensation. Due to the ideal distance between cationic groups, polyamines are perfect counterions for oligodeoxyribonucleotides. We have synthesized non-nucleosidic analogues built from units that carry different diol structures instead of sugar residues and functionalized with polyamines. The non-nucleosidic analogues were attached as internal or 5′-terminal modifications in oligodeoxyribonucleotide strands. The thermodynamic studies of these polyaminooligonucleotide analogues revealed stabilizing or destabilizing effects that depend on the linker or polyamine used.

Full text of DOI:10.3390/molecules200712652

Molecules 2015, 20, 12652-12669; doi:10.3390/molecules200712652
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Non-Nucleosidic Analogues of Polyaminonucleosides and
Their Influence on Thermodynamic Properties of
Derived Oligonucleotides
Jolanta Brzezinska and Wojciech T. Markiewicz *
Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14, 61-704 Poznań,
Poland; E-Mail: jabrzoza@ibch.poznan.pl
*
Author to whom correspondence should be addressed; E-Mail: markwt@ibch.poznan.pl;
Tel.: +48-61-852-8503 (ext. 180); Fax: +48-61-852-0532.
Academic Editors: Mahesh K. Lakshman and Fumi Nagatsugi
Received: 17 March 2015 / Accepted: 9 July 2015 / Published: 13 July 2015
Abstract: The rationale for the synthesis of cationic modified nucleosides is higher expected
nuclease resistance and potentially better cellular uptake due to an overall reduced negative
charge based on internal charge compensation. Due to the ideal distance between cationic
groups, polyamines are perfect counterions for oligodeoxyribonucleotides. We have synthesized
non-nucleosidic analogues built from units that carry different diol structures instead of sugar
residues and functionalized with polyamines. The non-nucleosidic analogues were attached as
internal or 5′-terminal modifications in oligodeoxyribonucleotide strands. The thermodynamic
studies of these polyaminooligonucleotide analogues revealed stabilizing or destabilizing
effects that depend on the linker or polyamine used.
Keywords: polyaminonucleoside analogue; non-nucleosidic analogue; spermine; putrescine;
oligonucleotide; duplex DNA; thermodynamic stability
1
. Introduction
The polyanionic character of nucleic acids and their synthetic fragments is a barrier for their
introduction into cells. Many laboratories try to improve nucleic acid delivery to cells by synthesis of
cationic bioconjugates [1–3], bioconjugate analogues with reduced polyanionic character [4–6], or a
great variety of polymeric transfection media [7–12]. On the other hand, there are studies to elucidate a

Molecules 2015, 20
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specific mode of small cationic molecular drugs interactions with biomolecules (e.g., nucleic acids,
proteins) useful in developing their more active analogues [13]. Among this clinically important class
of compounds are antibiotics carrying aminosugar residues [14].
Chemically modified oligonucleotides have been utilized as indispensable materials for DNA gene
therapy [15,16], gene regulation [17,18], chip technology [19,20] and recent nanotechnology [21–23]
because of their hybridization affinity for target DNA and/or RNA molecules [24]. The polyanionic
character of antisense and siRNA oligonucleotides is a major cause of insufficient cellular uptake and side
effects such as binding to serum proteins. The combination of nucleotides with aminoalkyl chains greatly
enhances the variety of possible structures as well as their potential application [24]. Thus, oligonucleotides
possessing cationic functionalities in addition to the anionic phosphate backbone have been shown to
exhibit promising properties [25–28]. Polyamines, putrescine, spermine and spermidine, are involved
in the regulation of gene function [29,30]. In vitro, they stabilize DNA and RNA duplexes [31,32],
especially ones with imperfect base pairing [33]. The distance between amino groups of three and four
carbon atoms is practically the same as the distance between phosphate anions in the backbone of
DNA making polyamines the perfect compounds for creating “zwitterionic” oligonucleotides [27]. It is
known that polyamines interact with DNA and RNA in different ways. The electrostatic binding is
performed via water molecules, by hydrogen bonding with polar functional groups or with hydrophobic
surfaces of nucleobases. There have been numerous studies aimed at determining these interactions
using NMR imaging, circular dichroism, Raman spectroscopy, IR spectroscopy, X-ray crystallography
and differential scanning calorimetry. In spite of these extensive studies, precise mechanisms for the
interaction between polyamine and DNA is still not fully understood in vivo [34].
The non-nucleosidic analogs of polyaminonucleosides have not been examined so far. Therefore, our
aim was to elaborate versatile procedures for synthesis of non-nucleosidic polyamine derivatives and
learn their properties within oligonucleotide chains. The choice of carbon chain skeletons of analogues
(
Figure 1) was based on the extent of their commercial availability.
O
HO
OCH3
O
O
HO
HO
OH
HO
O
OH
Figure 1. Substrates for the non-nucleosidic polyaminonucleoside analogues.
The obtained polyamine building blocks were incorporated at the 5′-end and internal positions
within an oligodeoxyribonucleotide chain and the resulting conjugates were evaluated for their
hybridization properties.
2
. Results and Discussion
2
.1. Chemistry
The synthesis of the non-nucleosidic polyamine derived phosphoramidite building blocks containing
different linkers is shown in Scheme 1.

Molecules 2015, 20
12654
O
O
O
O
O
O
O
DMTO
NHR
NHR
ii
iv
OH
N
H
N
H
1
R O
3 R = H
1
2
5
6
R = TFA, R = H
i
iii
v
4 R = TFA
1
1
bis-MPA
R = TFA, R = P(N-iPr )OCH CH CN
2
2
2
O
O
O
R
N
O
NHR
vi
O
N
N
R
O
O
H
H
H
7
(S) or 7a (R)
8
(S) or 8a (R) R = H
iii
vii
9 (S) or 9a (R) R= TFA
O
R
N
DMTO
NHR
N
N
R
1
R O
H
H
1
1
0 (S) or 10a (R) R = TFA, R = H
v
1
1
1 (S) or 11a (R) R = TFA, R = P(N-iPr )OCH CH CN
2
2
2
O
O
O
R
N
ix
2
HO
NHR
R O
N
N
R
OR2
H
2
2
1
1
2 R = H
14 R = H, R = TBDMS
viii
iii
2
2
3 R = TBDMS
x
15 R = TFA, R = TBDMS
O
R
DMTO
N
NHR
N
H
N
R
OR1
1
1
1
6 R = TFA, R = H
v
1
7 R = TFA, R = P(N-iPr )OCH CH CN
2
2
2
Scheme 1. Synthetic routes to non-nucleosidic polyaminonucleoside analogues: (i)
benzaldehyde, p-TsOH, DMF; (ii) SOCl , putrescine, Et N, DCM; (iii) (F CCO) O, pyridine;
iv) (a) 6 M HCl, reflux, (b) DMTCl, pyridine; (v) (iPr N) POCH CH CN, 5-ethylthio-1H-
tetrazole, DCM; (vi) spermine, MeOH; (vii) (a) p-TsOH, MeOH, (b) DMTCl, pyridine;
viii) TBDMSCl, DMAP, imidazole, CH CN; (ix) spermine, MW, 40 min; (x) (a)
TEAHF,THF/dioxane, (b) DMTCl, pyridine.
2
3
3
2
(
2
2
2
2
(
3
We decided to use 2,2-bis(hydroxymethyl)-propionic acid (bis-MPA, 1) that was earlier applied in
dendrimer synthesis [35,36].
Bis-MPA (1) was protected with benzaldehyde [37] as it is more lipophilic than a isopropylidene
group preferred to ease the final workup procedure and increase yield when using an excess of
polar polyamines [38,39]. The protection of hydroxyl groups in bis-MPA gives rise to formation of

Molecules 2015, 20
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two stereoisomers in the ratio ca 2:1, however, further reactions were carried out without separation of
isomers. Several approaches for activation of carboxyl function (2) were checked. Reactions with
1
1
,4-butylamine as a model amine in the presence of carbonyldiimidazole or 2-chloro-4,6-dimethoxy-
,3,5-triazine gave the expected n-butylamide derivative (results not shown). The best results were
obtained when activation was performed with thionyl chloride in the presence of pyridine and traces of
DMF. Thus, reaction with molar excess of putrescine (1,4-diaminobutane) at ambient conditions led to
N-(4-aminobutyl)-5-methyl-2-phenyl-1,3-dioxane-5-carboxyamide 2 in 60% yield (Scheme 1).
We also synthesized the analogs based on the enantiomers (7) and (7a) which were coupled with
excess of spermine in dry MeOH. Due to the lack of a chromophore in synthesized compounds, a
reaction control was performed using TLC plates stained with a solution of fluorescein (free acid) in
acetonitrile. Spermine derivatives (8, 8a) were isolated by several extractions with large amounts of
organic solvent and used in the next step without additional purification. The configuration of the
branching point in these linkers is retained throughout the synthetic procedure. After protection of
the amine function with trifluoroacetyl groups, 1,3-dioxolane was hydrolyzed to prepare polyamine
non-nucleosidic derivatives for selective 4,4′-dimethoxytritylation. In order to avoid transacetylation of
trifluoroacetyl groups during the isopropylidene cleavage of 9 we used p-TsOH instead acetic acid.
Otherwise, amino groups would be at least in part protected with Ac instead of TFA and their deprotection
with ammonia would not be possible under the conditions of the final deprotection of oligonucleotides.
Commercially available precursor α-hydroxy-γ-butyrolactone (12) was used as a starting material to
obtain a polyaminonucleoside analogue (17). This compound provides a three carbon distance between
the phosphate groups after introduction into the oligodeoxyribonucleotide strand. It also contains a
chiral carbon center with a secondary hydroxyl group and the carbonyl group corresponding to the
3
′-hydroxyl and 2′-carbon of nucleosidic residues, respectively. The lactone-spermine conjugate was
subsequently obtained after protection of the secondary hydroxyl group of the lactone (13) with lipophilic
and a bulky t-butyldimethylsilyl (TBDMS) group. Thus, this eased the isolation of the product (14)
resulting from the coupling reaction with spermine (Scheme 1). The reaction of protected lactone 13 and
unprotected spermine was performed by microwave synthesis to achieve the desired product 14 with
64% yield, even when both reagents were used in equimolar ratio. To eliminate side reactions during
the condensation step of DNA synthesis, the protection of putrescine and spermine amino functions
with trifluoroacetyl groups was ensured (as previously described [40]. For all non-nucleosidic analogue
yields of trifluoroacetylation ranged from 58% (spermine derivatives) up to 70% (putrescine derivative)
(Scheme 1). Conversion to the corresponding phosphoramidite derivative was preceded by removal
of protecting groups of hydroxyl function with HCl (4) and p-TsOH (9, 9a) acids or Et N*3HF (15).
3
The protection 5′-OH-groups with a 4,4′-dimethoxytrityl group was used in a last step of the 3′-OH
reaction with 2-cyanoethyl-N,N,N′,N′-tetraisopropylaminophosphane and 5-(ethylthio)-1H-tetrazole as
the activator. However, due to similar physical properties, the separation of the products from impurities
was difficult by silica gel column chromatography. This was resolved by precipitation from hexane.
The spermine derivatives of (R) and (S) enantiomers of glyceric acid gained an additional chiral center
as phosphoramidites (11, 11a). In the case of the polyamine derivative of α-hydroxy-γ-butyrolactone,
opening of the lactone ring 14 led to a mixture of enantiomers. Thus, after phosphitylation, compound
1
7 as a mixture of enantiomers was obtained. All amidites were lyophilized and were stable during
long-term storage at −20 °C.

Molecules 2015, 20
.2. Oligonucleotides
The phosphoramidites (6, 11, 11a, 17) were used for synthesis of polyamine analogs of
12656
2
oligodeoxyribonucleotides (Figure 2) using a 12-mer as a reference sequence (Table 1). The coupling
efficiencies of these phosphoramidites were 53%–70% as determined by measuring detritylation. The
modified oligodeoxyribonucleotides were obtained after a standard deprotection procedure, and their
structures were confirmed by the MALDI-TOF (Table 1). The oligodeoxyribonucleotides ON5–ON6
and ON9 were used as pure enantiomers and ON4 and ON8 as mixtures of diastereoisomers.
Table 1. Sequences of oligodeoxyribonucleotides and MALDI-TOF MS data.
m/z [M − H]−
No.
Sequence 5′ → 3′ a
Calcd
Found
ON1
ON2
CTC AAG CAA GCT
CTC ACA TGC GCG
3614.42 3613.08
3606.40 3605.54
ON3 X0 CTC AAG CAA GCT 3994.42 3993.19
ON4 X1 CTC ACA TGC GCG 3973.12 3972.23
ON5 X2 CTC ACA TGC GCG 3959.54 3959.24
ON6 X3 CTC ACA TGC GCG 3959.54 3959.57
ON7 CTC AAG X0 CAA GCT 3994.42 3993.25
ON8 CTC ACA X1 TGC GCG 3973.12 3973.12
ON9 CTC ACA X2 TGC GCG 3959.54 3957.45
a
R = H for X0–X3 in ON3–ON6 and R = 3′-end oligo for X0–X2 in ON7–ON9.
O
RO
NH2
X0
N
R1O
H
OR
O
O
H
N
X1
X2
NH2
N
H
N
H
R1O
RO
H
N
NH2
N
H
N
H
R1O H
RO
O
H
N
NH2
X3
N
H
N
H
R1O H
R = H or 3'-end oligo
1
R = 5'-end oligo
Figure 2. Non-nucleosidic polyamino-nucleoside analogues units in oligodeoxyribonucleotide strands.

Molecules 2015, 20
.3. Stability of Duplexes with Modified Oligodeoxyribonucleotides
The influence of conjugated polyamines on the stability of the DNA duplexes was studied using
12657
2
UV melting spectra. It was shown previously that polyamine conjugation to oligodeoxyribonucleotides
results in stabilizing of DNA duplexes and triplexes [2–4,38,39,41–43]. Recently, we described the
NMR structure of a DNA duplex carrying a single spermine modified deoxycytidine unit [34]. This
Sp
modification moderately stabilizes the DNA duplex (Figure 3, dC vs. RF1) and does not perturb the
DNA structure.
Figure 3. Changes in free energy (ΔG) of polyamine modified duplexes: RF-RF3-
Sp
reference duplexes, dCSp—spermine modified (dC = 4-N-[4,9,13-triazatridecan-1-yl]-2′-
deoxycytidine) duplex [34]; ON3, ON7—non-nucleosidic putrescine analogues (Table 2);
ON4–ON6 and ON8–ON9—non-nucleosidic spermine analogues (Table 2).
The 5′-end dangling modifications in oligodeoxyribonucleotide duplexes usually increase duplex
stability and do not show large sequence dependence [44,45]. Since the terminal unpaired nucleotides are
not involved in base pairing, stacking, electrostatic, perhaps to some extent, hydrophobic interactions are
responsible for the thermodynamic effects of the 5′- and 3′-dangling ends. The nearest-neighborhood
model assumes the duplex region for a dangling end to have the same calculated thermodynamic stability
as a matching blunt-end duplex [32,33].
Thus, the stability was increased when X1 and X3 (Scheme 1) were incorporated at the 5′ position
(Table 1, entry ON4 and ON6), but for incorporations of X0 and X2 (Scheme 1) a moderate destabilizing

Molecules 2015, 20
12658
effect was observed (Table 1, entry ON3 and ON5). Despite lack of a nucleobase and higher
conformational flexibility at the 5′-end of the strand the observed stabilities are rather typical for 5′-end
dangling units as described in the literature. Thermodynamic data (Table 2, Figure 1) show that X3
results in ΔΔG (−0.3 kcal/mol) similar to unpaired nucleotide (−0.1 to −0.5 kcal/mol) at the 5'-end. The
change of ΔG for X1 is even higher (ΔΔG = −0.64 kcal/mol). These results suggest that the lack of a
nucleobase does not affect the thermal stability of the duplexes. This might indicate that increased
duplex stability is provided by the three positive charges in the spermine chain. In the case of the
putrescine residue, X0, which introduces one positive charge only, a small decrease of duplex stability
was observed. (Figure 1, ON3). One can conclude that the small decrease in duplex stability observed
for ON5 (Figure 1) containing spermine attached to the glycerol isomer (X2) is caused by the structure
and configuration of this particular linker.
Table 2. DNA duplex stability of non-nucleosidic polyamine-modified oligodeoxyribonucleotides a.
−
1
Average of Curve Fits
T
M
vs. log (C
T
/4) Plots
TM
ΔTM b
Oligo
−ΔH°
−ΔS°
−ΔG°37
TM
−ΔH°
−ΔS°
[eu]
−ΔG°37
ΔΔG°37
[kcal/mol]
[eu]
[kcal/mol] [°C] [kcal/mol]
[kcal/mol] [°C] [kcal/mol] [°C]
A. Thermodynamic parameters of references duplexes
ON1 c 67.8 ± 9.0 185.6 ± 27.8 10.27 ± 0.44 55.1 72.6 ± 2.0 200.6 ± 6.2 10.45 ± 0.08 54.7
ON2 d 115.9 ± 4.8 322.1 ± 14.2 16.00 ± 041 64.6 108.5 ± 1.8 300.1 ± 5.4 15.46 ± 0.12 64.8
B. Thermodynamic parameters of duplexes with the polyamine analog as a dangling end
ON3 c 63.3 ± 6.8 171.5 ± 20.9 10.12 ± 0.37 55.7 69.09 ± 3.1 189.4 ± 9.7 10.33 ± 0.13 55.0
ON4 d 131.8 ± 10.4 368.8 ± 31.0 17.47 ± 0.80 65.1 114.0 ± 8.3 315.9 ± 24.9 16.10 ± 0.64 65.4
ON5 d 97.8 ± 9.4 268.4 ± 28.1 14.57 ± 0.68 64.8 98.9 ± 5.1 271.9 ± 15.4 14.59 ± 0.36 64.5
ON6 d 106.1 ± 4.7 292.4 ± 14.0 15.43 ± .0.37 65.4 110.9 ± 3.9 307.0 ± 11.7 15.76 ± 0.27 65.1
C. Thermodynamic parameters of duplexes with the polyamine analog as a bulge
-
-
-
-
+0.12
−0.64
+0.87
−0.3
−0.3
+0.6
−0.3
+0.3
ON7 c 62.7 ± 16.7 178.2 ± 53.8 7.50 ± 0.20 41.9 59.0 ± 4.2 166.5 ± 13.7 7.38 ± 0.08 41.5
ON8 d 71.6 ± 6.0 194.4 ± 18.3 11.31 ± 0.35 59.2 82.0 ± 2..0 226.3 ± 6.2 11.83 ± 0.10 58.4
ON9 d 81.2 ± 11.5 225.0 ± 35.6 11.45 ± 0.48 57.0 70 ± 6.4 192.1 ± 20.0 11.03 ± 0.25 58.1
+3.07
+3.63
+4.43
−13.2
−6.4
−6.7
a
b
−4
2
Solutions are 100 mM NaCl, 20 mM sodium cacodylate, 0.5 mM Na EDTA, pH 7; Calculated for 10 M total strand concentration;
c
d
Complementary strand 5′-AGC TTG CTT GAG-3′; Complementary strand 5′-CGC GCA TGT GAG-3′.
Next, we investigated the influence of polyamine derivatives X0, X1 and X2 on duplex stability,
inserted as bulges in the middle of the sequence (Table 1, entry ON7–ON9). The changes of duplex
stability caused by single nucleotide bulges differ and depend on the type of flanking bases [46,47].
Incorporation of X0–X2 resulted in a lowering of melting temperature independently of polyamine
residue. However, the melting data suggest that the duplex formation occurs with the proper W-C base
pairing despite of a slight increase in free energy for ON7–ON9 (Table 2, ΔΔG ca. 3–4.5 kcal/mol).
Moreover, the effect in ON7 where the putrescine bulge (X0) is flanked by GC/CG pairs is practically
the same as for ON8 and ON9 (spermine bulge, X1 and X2) flanked by AT/TA pairs. This can be
attributed to the stronger stabilizing effect of a spermine residue when compared to putrescine—three
positive charges vs. one. Yet, even three positive charges of a spermine residue do not neutralize the
bulge effect as such. The observed changes of ΔG for the studied duplexes (ON7–ON9) are in the range
observed for duplexes with a single bulge (ΔΔG, 2–6 kcal/mol) [46,47].

Molecules 2015, 20
12659
Linkers X1 and X2 differ in length by one carbon and this additional carbon in X1 makes this linker
somehow less rigid, allowing for more favorable placement of the polyamine residue. Thus, the energy
gain of 1.5 kcal/mol in ON4 when compared to ON5. Therefore, ON4 duplex is more stable than the
unmodified duplex. When the same modified linkers (X1 and X2) are inserted as bulges in a more
spatially “demanding environment”, the difference of free energies ΔΔG°37 of ON8 (+3.63) and ON9
(+4.43) is smaller (0.8 kcal/mol). The influence of linker structure is less profound when the modification
is inserted in the middle of chain.
Linkers X2 and X3 carrying a spermine residue differ only in the configuration of carbon (S and R
respectively). Comparison of ΔΔG°37 of ON6 (−0.3 kcal/mol) and ON5 (+0.87 kcal/mol) suggests that
X3 allows more favorable placement of polyamine residue This seems to indicate that in this case the
spermine residue attached to a glycerol linker with R-configuration (X3) is closer to the duplex
charged surface.
We would like to conclude that the overall effect of DNA modification with polyamine analogues
seems to offer a convenient way to modify nucleic acids properties. An attachment of polyamine residues
via various open chain linkers allows to maintain the general scheme of base pairing. Moreover, the
structure and configuration of the linkers seems to have a higher influence on stability when placed at
the 5′-end of DNA duplexes.
3. Experimental Section
3.1. General Methods
All reagents were of analytical grade, obtained from commercial resources and used without further
purification. For synthesis, solvents with quality pro analysis were used. Solvents were dried and distilled
following standard methods and kept over molecular sieve. All reactions were carried at room temperature
unless described otherwise. Column chromatography was performed with silica gel (Merck KGaA,
Darmstadt, Germany, 200–630 mesh) and TLC was carried out on precoated plates (Merck silica gel 60,
1
13
F
254). All NMR spectra were recorded at 298 K on Bruker AVANCE II (400 MHz, H, C) and Varian
3
1
Unity (300 MHz, P) spectrometers (Bruker BioSpin GmbH, Rheinstetten, Germany and Varian, Inc.,
Palo Alto, CA, USA). Chemical shifts (δ) are reported in parts per million (ppm). J values are given in
Hz. Mass spectra were recorded on the MicroTofQ mass spectrometer with electrospray ionization (ESI)
sources (ESI source voltage of 3.2 kV, nebulization with nitrogen at 0.4 bar, dry gas flow of 4.0 L/min at
temperature 220 °C) and Bruker Autoflex MALDI-TOF (Bruker Daltonik GmbH, Bremen, Germany).
Microwave reactions was performed in domestic microwave oven (800 W, Amica, Wronki, Poland).
Some NMR (Figures S1–S16) and MS (Figures S17–S22) spectra are available in Supplementary.
3.2. Synthesis of Monomers
Benzylidene-2,2-bis(oxymethyl)propionic acid (2) [37]. Benzaldehyde (4.2 mL, 40.7 mmol) was added
to a well-stirred solution of 2,2-bis(hydroxymethyl)-propionic acid (1) (5 g, 37 mmol) in DMF (30 mL),
followed by catalytic p-TsOH (0.355 g, 1.85 mmol) with stirring at room temperature for 4 days. The
reaction was quenched with NH
4
OH/EtOH (1 mL, 1:1). The solvent was evaporated and the residue
(2 × 100 mL). Organic extracts were dried
dissolved in DCM (100 mL) and washed with NaHCO
3

Molecules 2015, 20
12660
over anhydrous MgSO
4
and filtered then evaporated under reduced pressure. The residue was purified by
1
recrystallization from DCM to obtain product 2 as mixture of two isomers (77%). H-NMR (CDCl
3
);
CCO, 3.67 (d,
); isomer B: 7.28–7.51 (m, 5H, Ph), 5.41 (s, 1H, PhCHO ),
): 178.89
isomer A: 7.28–7.46 (m, 5H, Ph), 5.45 (s, 1H, PhCHO
2
), 4.65 (d, 2H, J = 11.7 Hz, OCH
2
2
H, J = 11.7 Hz, OCH
2
CCO), 1.02 (s, 3H, CH
3
2
13
4.14–4.06 (q, 4H, J = 11.23 Hz, J = 9.27 Hz, 2CH
2
), 1.57 (s, 3H, CH
3
). C-NMR (CDCl
3
(
C=O), 137.58 (C, Ph), 129.08 (CH, Ph), 128.23 (CH, Ph), 128.23 (CH, Ph), 101.86 (CH), 73.43 (CH
2
),
−
4
2.17 (CMe), 17.78 (CH
3
). MS (ESI) m/z: calcd for C12
H
14
O
4
221.0819 [M − H] , found 221.182.
N-(4-Aminobutyl)-5-methyl-2-phenyl-1,3-dioxane-5-carboxamide (3). Thionyl chloride (1.26 mL,
1
1
7.8 mmol) was added dropwise to ice-cooled solution of 2 (2 g, 8.9 mmol) in 45 mL DCM containing
0% of pyridine and 3 drops of DMF. The mixture was stirred for 2 h at room temperature, and the
excess thionyl chloride was removed by several co-evaporations with a mixture of DCM and toluene.
The brown residue was dissolved in DCM (46 mL) and the temperature was lowered to −10 °C.
Putrescine (4.47 mL, 44.5 mmol) and triethylamine (1.4 mL) in DCM (2 mL) was added after 15 min.
The mixture was stirred for 1h at room temperature, and the reaction was quenched with saturated
aqueous NaHCO
organic extracts were dried over anhydrous MgSO
chromatography with 4% MeOH in DCM as the eluent to give 3 (0.910 g, 60% yield) as a yellow oil.
1H-NMR (CDCl
): 7.34–7.43 (m, 5H, Ph), 7.03 (t, 1H, NHCO), 5.48 (s, 1H, PhCHO ), 4.33 (d, 2H,
J = 12 Hz, OCH CCO), 3.78 (d, 2H, J = 12 Hz, OCH CCO), 3.33 (q, 2H, J = 6.3 Hz, J = 5.8 Hz,
CONHCH ), 3.21 (q, 2H, J = 6.3 Hz, J = 5.8 Hz, NH CH , putrescine), 1.5–1.56 (m, 4H, 2 × CH
putrescine), 1.04 (s, 3H, CH ): δ (ppm) 178.89 (C=O), 137.45 (C, Ph), 128.7 (CH,
Ph), 128.03 (CH, Ph), 101.22 (CH), 75.07 (CH
putrescine), 18.03 (CH ); MS (ESI) m/z: calcd for C16
3
(3 mL). The mixture was extracted with DCM (2 × 100 mL). The combined
4
and concentrated under vacuum and purified by
3
2
2
2
2
2
2
2
,
1
3
3
); C-NMR (CDCl
3
2
), 47.6 (CMe), 47.6 (CH
2
, putrescine), 26.53 (CH
2
,
+
3
H
24
N
2
O
3
293.186 [M + H] , found 293.2037.
5
-Methyl-2-phenyl-N-(4-(2,2,2-trifluoroacetamid)butyl)-1,3-dioxane-5-carboxamide (4) 3 (0.430 g,
1.47 mmol) was co-evaporated with pyridine (3 × 5 mL), dissolved in pyridine (15 mL), followed by
the addition of N-methylimidazole (0.16 mL, 1.46 mmol). Then, trifluoroacetic anhydride (0.593 mL,
.41 mmol) was added dropwise to the mixture cooled at 0 °C. The mixture was stirred for 30 min and
then poured into saturated aqueous NaHCO (30 mL), and extracted with DCM (3 × 50 mL). The
combined organic extracts were dried over anhydrous Na SO and concentrated under vacuum. The
4
3
2
4
residue was purified by chromatography using DCM as the eluent to give 4 (0.500 g, 52% yield) as an
1
oil. H-NMR (CDCl
3
): 7.33–7.45 (m, 5H, Ph), 7.03 (t, 1H, NHCO), 5.5 (s, 1H, PhCHO
CCO), 3.78 (d, 2H, J = 11.7 Hz, OCH CCO), 3.2–3.4 (m, 4H, 2NHCH
). C-NMR (CDCl ): 182.83 (C=O), 158.9 (C=O), 137.45
C, Ph), 128.7 (CH, Ph), 128.03 (CH, Ph), 125.53 (C, CF ), 101.22 (CH), 75.07 (CH ), 47.4 (CMe),
5.3 (CH , putrescine), 26.53 (CH , putrescine), 18.03 (CH ); F NMR 1.78 (s, 3F, CF ); MS (ESI)
m/z: calcd for C18 389.1683 [M + H] , found 389.1627.
2
), 4.38 (d, 2H,
J = 11.7 Hz, OCH
2
2
2
), 1.49–1.54
1
3
(
m, 4H, CH
2
, putrescine), 1.06 (s, 3H, CH
3
3
(
3
2
1
9
4
2
2
3
3
+
H
23
F
3
N
2
O
4
3
-(4,4′-Dimethoxytrityl)-2-(hydroxymethyl)-2-methyl-N-(4-(2,2,2-trifluoroacetamido)butyl)propanamide
5). 4 (0.240 g, 0.61 mmol) was dissolved in a mixture conc. aq HCl/EtOH (1:5, v/v) and refluxed for
2 h. The excess of HCl was removed by several co-evaporations with a mixture of methanol and
(
7

Molecules 2015, 20
12661
toluene and finally a brown oil with was dried by co-evaporation with anhydrous pyridine (2 × 5 mL)
and dissolved in anhydrous pyridine (2.4 mL). To this solution 4,4′-dimethoxytrityl chloride (0.243 g,
0
.72 mmol) was added and the reaction was quenched after 3 h by adding saturated aqueous NaHCO
The resulting solution was extracted with DCM and the combined organic extracts were washed with
brine, dried over Na SO and concentrated under vacuum. The residual yellow oil was purified by
chromatography with 5% MeOH in DCM as the eluent to give 0.18 g (49%) 5 as a white foam.
1H-NMR (CDCl
): δ (ppm) 7.42–7.2 (m, 10H, Ph, NHCO), 7.04 (t, 1H, NHCO), 6.89 (m, 4H, Ph),
.82 (s, 3H, OCH ), 3.83–3.62 (m, 4H, OCH CCO), 3.43–3.23 (m, 4H, 2NHCH ), 1.4–1.8 (m, 4H,
CH , putrescine), 1.2 (s, 3H, CH ). C-NMR (CDCl ): δ (ppm) 174.4 (C=O), 158.5 (2C, COCH ),
58.4 (C=O, TFA), 144.6 (C, Ph), 135.5 (C, Ph), 130.0 (CH, Ph), 128.3 (CH, Ph), 128.0 (CH, Ph),
27.8 (CH, Ph), 126.8 (C, CF ), 113.1 (CH, Ph), 86.2 (C), 66.9 (CH ), 65.5 (CH ), 55.1 (CH ), 47.5 (C,
, putrescine), 18.7 (CH ); MS (ESI) m/z: calcd for
3
.
2
4
3
3
2
1
1
3
2
2
1
3
2
3
3
3
3
2
2
3
CMe), 43.2 (CH
2
, putrescine), 24.5 (CH
2
3
+
C
32
H
37
F
3
N O
2 6
603.2676 [M + H] , found 603.261.
3-[(4,4′-Dimethoxytrityl)-2-(hydroxymethyl)-2-methyl-N-(4-(2,2,2-trifluoroacetamido)butyl)propanamide]
phosphoramidite (6). 2-Cyanoethyl-N,N,N,N-tetraisopropylphosphoramidite (0.9 mL, 0.3 mmol) was
added to the solution of 5 (0.143 g, 0.234 mmol) and 5-(ethylthio)-1H-tetrazole (0.0273 g, 0.21 mmol)
in dichloromethane (1.2 mL) and the mixture was stirred at room temperature. After 2 h, TLC revealed
complete reaction. The mixture was diluted with dichloromethane, washed with saturated sodium
bicarbonate solution and the organic extracts were dried over Na
2
SO
4
. The product was purified by
silica gel chromatography with benzene–Et N (10%) and lyophilisation from benzene to give 6 (134 mg,
3
3
1
7
0% yield) as a white powder. P-NMR (C
6
H ): δ (ppm) 148.30; 148.54. MS (ESI) m/z: calcd for,
6
+
C
41
H
54
F
3
N O
4 7
P [M + H] 803.3755, found 803.371.
(
S)-N-(4,9,13-Triazatridecan-1-yl)-2,2-dimethyl-1,3-dioxolane-4-carboxyamide (8). The methyl
S)-2,2-dimethyl-1,3-dioxalane-4-carboxylate (7) (0.45 mL, 3 mmol), spermine (2.4 g, 12 mmol) were
(
dissolved in anhydrous methanol (1 mL) and the resulting solution was stirred at room temperature for
8–72 h at 25–35 °C. The solvent was evaporated under reduced pressure and the residue was purified
4
by column chromatography over silica gel (MeOH/MeNH
2
/H O) to give 8 (720 mg, 70% yield) as
2
1
yellow oil. H-NMR (CDCl
3
): δ (ppm) 7.22 (m, 1H, NHCO), 4.45 (q, 1H, J = 5.37 Hz, J = 2.44 Hz,
COCH), 4.25 (t, 1H, J =7.8, OCHCO), 4.05 (q, 1H, J = 5.37 Hz, J = 3.41 Hz, COCH), 3.38–3.24 (m,
H, CONHCH ), 2.53–2.65 (m, 6H, CH ), 2.05 (2H, NH), 1.66 (m, 2H, spermine), 1.49 (m, 2H,
spermine), 1.44 (s, 3H, CH ), 1.44 (s, 3H, CH
4.8 (C), 67.6 (C), 49.1 (CH ), 46.8 (CH
CH ). MS (ESI) m/z: calcd for C16
2
2
2
13
3
3
); C-NMR (CDCl
3
): δ (ppm) 171.3 (C=O), 110.6 (OCO),
7
2
2
), 37.1 (CH
2
), 28.6 (CH ), 27.1 (CH ), 26.0 (CH ), 24.8
2
2
2
+
(
3
H
34
N
4
O
3
[M + K] 369.2262, found 370.2801.
(R)-N-(4,9,13-Triazatridecan-1-yl)-2,2-dimethyl-1,3-dioxolane-4-carboxyamide (8a). The methyl
(
R)-2,2-dimethyl-1,3-dioxalane-4-carboxylate (7a) (0.45 mL, 3 mmol) was converted into compound 8a
1
following the above procedure (566 mg, 55% yield). H-NMR (CDCl
3
): δ (ppm) 7.19 (m, 1H, NHCO),
4
.45 (q, 1H, J = 5.37 Hz, J = 2.44 Hz, COCH), 4.26 (t, 1H, J = 7.8 Hz, OCHCO), 4.05 (q, 1H,
J = 5.37 Hz, J = 3.41 Hz, COCH), 3.43–3.3 (m, 2H, CONHCH
2
), 2.74–2.59 (m, 6H, CH
2
), 1.76 (m,
13
3
2H, spermine), 1.63 (m, 2H, spermine), 1.47 (s, 3H, CH ), 1.37 (s, 3H, CH
3
); C-NMR (CDCl ): δ
3

Molecules 2015, 20
12662
(
ppm) 171.3 (C=O), 110.6 (OCO), 74.8 (C), 67.6 (C), 49.1 (CH
7.1 (CH ), 26.0 (CH ), 24.8 (CH ). MS (ESI) m/z: calcd for C16
found 331.2701.
2
), 46.8 (CH
2
), 37.1 (CH
2
), 28.6 (CH
2
),
+
2
2
2
3
H
34
N
4
O
3
[M + H] 331.2704,
(S)-N-[Tris(2,2,2-trifluoroacet-1-yl)-4,9,13-triazatridecane]-2,2-dimethyl-1,3-dioxolane-4-carboxyamide (9).
8
(0.200 g, 0.6 mmol) was co-evaporated with anhydrous pyridine (3 × 5 mL) and redissolved in
pyridine (6 mL). Trifluoroacetic anhydride (0.5 mL, 3.6 mmol) was added dropwise to a cooled and
stirred solution at 0 °C. The solution was warmed to room temperature and stirred for 30 min, quenched
with NaHCO
aqueous NaCl, dried over Na
chromatography with 2% MeOH in DCM as the eluent to give 9 (0.2 g, 54% yield) as a white foam.
1H-NMR (CDCl
): δ (ppm) 7.09 (m, 1H, NHCO), 6.72 (m, 1H, NHCO), 4.46 (q, 1H, J = 5.34 Hz,
J = 3.78 Hz, COCH), 4.07 (q, 1H, J = 5.34 Hz, J = 3.78 Hz, COCH), 4.28 (t, 1H, J = 7.64 Hz,
OCHCO), 3.49–3.25 (m, 8H, NHCH CH , spermine), 1.86–1.78 (m, 2H, spermine), 1.61 (m, 2H,
3
and extracted with CH
2
Cl . The combined organic extracts were washed with saturated
2
2
SO and concentrated under vacuum. The residual oil was purified by
4
3
2
2
1
9
spermine), 1.49 (s, 3H, CH
calcd for C22
3
), 1.39 (s, 3H, CH
3
). F-NMR: 6.98–7.22 (m, 9F, 3CF ). MS (ESI) m/z:
3
−
H
30
F
9
N
4
O
6
[M − H] 617.2022, found 617.3021.
(R)-N-[Tris(2,2,2-trifluoroacet-1-yl)-4,9,13-triazatridecane]-2,2-dimethyl-1,3-dioxolane-4-carboxyamide
(
9a). 8a (0.350 g, 1.05 mmol) was converted into 9a (0.368 g, 58% yield) following procedure for 9.
1H-NMR (CDCl
): δ (ppm) 7.17 (m, 1H, NHCO), 6.76 (m, 1H, NHCO), 4.47 (q, 1H, J = 7.2 Hz,
J = 3.3 Hz, COCH), 4.07 (m, 1H, COCH), 4.29 (t, 1H, J = 8 Hz, OCHCO), 3.48–3.26 (m, 8H,
NHCH CH , spermine), 1.88–1.82 (m, 2H, spermine), 1.62 (m, 2H, spermine), 1.49 (s, 3H, CH ), 1.39
s, 3H, CH
[M + K]+
3
2
2
3
1
9
(
3
). F-NMR: 6.98–7.22 (m, 9F, 3CF
3
). MS (ESI) m/z calcd for C22
H
31
F
9
N
4
O
3
6
57.1737, found 657.175.
(
S)-N-[Tris(2,2,2-trifluoroacet-1-yl)-4,9,13-triazatridecane]-3-(4,4′-dimethoxytrityl)-3-hydroxypropanamide
10). To a solution of 9 (0.500 g, 0.75 mmol) in MeOH (4 mL), p-toluenosulfonic acid (0.03 g,
.15 mmol) was added at room temperature. The mixture was stirred for ca 30 h at room temperature,
and solvent was evaporated. The mixture was diluted with ethyl acetate and washed with aq. saturated
NaHCO . The combined organic extracts were dried over Na SO and concentrated under vacuum.
The resulting crude was co-evaporated with pyridine (4 × 5 mL) and dissolved in pyridine (2.5 mL).
,4′-Dimethoxytrityl chloride (0.330 g, 0.975 mmol) was added in portions at room temperature. The
mixture was stirred for 3 h at room temperature, and the reaction was quenched with methanol (1 mL)
and saturated aq. NaHCO (10 mL). The mixture was extracted with DCM. The combined organic
extracts were dried over MgSO and concentrated under vacuum. The resulting orange oil was purified
by chromatography with 2%–3% MeOH in CH Cl (with 0.2% of pyridine by vol.) as the eluent to
give 10 (0.494 g, 69% yield) as a white solid. H-NMR (CDCl ): δ (ppm) 7.39–6.8 (m, 15H, DMT,
NHCO), 4.19–4.13 (m, 1H, OCHCO), 3.75 (s, 6H, 2OCH ), 3.51–3.22 (m, 14H), 1.99–175 (m, 4H,
): δ (ppm) 171.9 (C), 171.7 (C), 158.7 (C),
44.4 (C), 135 (C), 129.9 (CH), 127,9 (CH), 127 (CH), 125.8 (C), 121 (C), 113.2 (CH), 86.8 (C), 77.2
CH), 67.7 (CH ), 55.2 (CH ), 46.3 (CH ), 36.1 (CH ), 35.1 (CH ), 29.7 (CH ), 27.1 (CH ). MS (ESI)
(
0
3
2
4
4
3
4
2
2
1
3
3
1
3
spermine), 1.68–1.55 (m, 4H, spermine). C-NMR (CDCl
3
1
(
2
3
2
2
2
2
2

Molecules 2015, 20
12663
+
[M + K]+
m/z: calcd for C40H F N O
45 9 4 8
[M + Na] 903.2991, found 903.2938; calcd for C40
H F N O
45 9 4 8
9
19.2731, found 919.266.
(R)-N-[Tris(2,2,2-trifluoroacet-1-yl)-4,9,13-triazatridecane]-3-(4,4′-dimethoxytrityl)-3-hydroxypropanamide
(
10a). 9a (0.350 g, 1.05 mmol) was converted into 10a (0.383 g, 54% yield) following procedure for
1
1
0. H-NMR (CDCl
3
): δ (ppm) 7.39–6.8 (m, 15H, DMT, NHCO), 4.2–4.12 (m, 1H, OCHCO), 3.75
(
s, 6H, 2OCH
3
), 3.48–3.18 (m, 14H), 1.91–1.74 (m, 4H, spermine), 1.63–1.51 (m, 4H, spermine). MS
−
(
ESI) m/z: calcd for C40
H
45
F
9
N
4
O
8
[M − H] 879.3015, found 879.3028.
2
-(S)-[(4,4′-Dimethoxytrityl)-3-(hydroxymethyl)-N-((2,2,2-trifluoroacet-1-yl)-4,9,13-triazatridecane)
propanamide]phosphoramidite (11). The compound 10 was converted into compound 11 following
procedure for 6. Except purification: crude phosphoramidite was trice precipitated from hexane and
31
finally lyophilized from benzene to give 11 (0.210 g, 52% yield) as a white light solid. P-NMR
(
C
6
H
6
): δ (ppm) 151.89; 151.68; 148.99; 148.68; 148.56; 146.81. MS (ESI) m/z: calcd for
+
O
P [M + K]+
C
49
H
62
F
9
N
6
NaO
9
P [M + Na] 1103.4070, found 1103.4575; calcd for C49
H
62
F
9
KN
6
9
found 1119.4509.
2
-(R)-[(4,4′-Dimethoxytrityl)-3-(hydroxymethyl)-N-((2,2,2-trifluoroacet-1-yl)-4,9,13-triazatridecane)
propanamide]phosphoramidite (11a). 10a (0.200 g, 0.226 mmol) was converted into compound 11a
1
(
0.128 g, 52% yield) according to procedure for 11. H-NMR (CDCl
3
): δ (ppm) 7.14–7.38 (m, 9H, DMT),
), 3.12–3.55 (m,
CN), 1.41–1.92 (m, 8H, spermine)
6
1
1
.78 (m, 4H, DMT), 4.8 (m, 1H, COCH), 4.65 (m, 1H, OCHCO), 3.73 (s, 6H, 2OCH
6H, CH OH, spermine, CH OP) 2.4–2.78 (m, 5H, 2CH, iPr, CH
.17–1.23 (m, 12H, iPr). P-NMR (C
3
2
2
2
3
1
6
H
6
): δ (ppm) 151.76; 151.42; 149.24; 149.05; 148.96; 148.83.
−
MS (ESI) m/z: calcd for C49
H
62
F
9
N O
6 9
P [M − H] 1079.4094, found 1079.63.
3
-(tert-Butyldimethylsilyloxy)butyrolactone (13) [48]. tert-Butyldimethylchlorosilane (1.50 g, 9.63 mmol),
imidazole (0.470 g, 3.21 mmol) and N,N-dimethylaminopyridine (0.392 g, 3.21 mmol) were added to a
solution of α-hydroxy-γ-butyrolactone (12) (0.25 mL, 3.21 mmol) in anhydrous CH CN (16 mL). The
reaction mixture was stirred at room temperature for 16 h. Then, solvent was evaporated under reduced
pressure. The residue was partitioned between saturated aq. NaHCO and Et O (3 × 50 mL). The
organic extracts were washed with brine, dried over MgSO . The crude residue was purified by column
O 4:1→2:1) to give compound 12 as colourless oil (0.485 g, 70%
O), 4.15–4.2 (dt, 1H, OCH), 2.16–2.5 (m, 1H,
3
3
2
4
chromatography (eluent hexane/Et
2
1
yield). H-NMR (CDCl
3
): δ (ppm) 4.35–4.4 (m, 2H, CH
), 0.9 (s, 9H, 3CH , t-Bu), 0.16 (s, 3H, CH CSi), 0.14 (s, 3H, CH
δ (ppm) 175.86 (C=O), 68.19 (CH), 64.72 (CH
CH ); MS (ESI) m/z: calcd for C10
2
1
3
OCHCH
2
3
3
3
CSi); C-NMR (CDCl
3
):
2
), 33.3 (CH
2
), 25.61 (CH
3
), 18.19 (SiC(CH ), 5.5
3 3
)
−
(
3
H
20
O
3
Si [M − H] 215.1103, found 215.0295.
N-(4,9,13-Triazatridecan-1-yl)-2-(tert-butylodimethylsiloxy)-butyramide (14). A solution of 13 (0.216 g,
mmol) and spermine (0.202 g, 1 mmol) in a small (10 mL) Erlenmeyer flask was kept in microwave
oven for 45 min. The flask was cooled down and the mixture was diluted with DCM (20 mL), washed
with NaHCO (3 × 30 mL), brine (30 mL), and organic layers dried over MgSO . After evaporation, the
as the eluent to give 14 (0.266 g, 64% yield) as
1
3
4
residue was chromatographed using MeOH/H
2
O/MeNH
2
1
oil. H-NMR (CDCl
3
): δ (ppm) 7.02 (t, 1H, NHCO), 4.25 (t, 1H, J = 6.01 Hz, COCHOSi), 3.71 (q, 2H,

Molecules 2015, 20
12664
),
J = 5.89 CH
2
OH), 3.38–3.32 (m, 2H, spermine), 2.65–2.58 (m, 4H), 1.99–1.94 (m, 2H, OCHCH
, t-Bu), 0.11 (s, 3H, CH CSi), 0.08
Si [M + H] 419.3417, found 419.3539.
2
1
.76 (m, 2H, spermine), 1.63 (m, 2H, spermine), 0.91 (s, 9H, 3CH
3
3
+
(
s, 3H, CH
3
CSi). MS (ESI) m/z: calcd for C20
H
47
N
4
O
3
N-[Tris(2,2,2-trifluoroacet-1-yl)-4,9,13-triazatridecane]-2-(tert-butyldimethylsiloxy)-butyramide (15).
4 (0.266 g, 0.64 mmol) was co-evaporated with anhydrous pyridine (3 × 5 mL) and redissolved in
pyridine (6 mL). Trifluoroacetic anhydride (0.517 mL, 3.83 mmol) was added dropwise at 0 °C. The
solution was warmed to room temperature and stirred for 30 min., quenched with NaHCO and
extracted with DCM (3 × 30mL). The combined organic extracts were washed with brine, dried over
1
3
MgSO
4
and purified by chromatography with 2%–3% MeOH in DCM as the eluent to give 15 (0.297 g,
1
6
1
2
6% yield) as an oil. H-NMR (CDCl
3
): δ (ppm) 7.09 (bs, 1H, NHCO), 6.83 (bs, 1H, NHCO), 4.27 (t,
OH), 3.47–3.15 (m, 6H), 2.01–1.75 (m, 4H), 1.63 (m,
H, COCHOSi), 3.73 (q, 2H, J = 5.89 Hz, CH
H, spermine), 0.93 (s, 9H, 3CH , t-Bu), 0.12 (s, 3H, CH
). MS (ESI) m/z: calcd for C26
2
19
3
3
CSi), 0.10 (s, 3H, CH
3
CSi). F-NMR 6.98–7.22
−
(m, 9F, CF
3
H
43
F
9
N
4
O
6
Si [M − H] 705.2730, found 705.2809.
N-[Tris(2,2,2-trifluoroacet-1-yl)-4,9,13-triazatridecane]-4-(4,4′-dimethoxytrityl)-butyramide
(16).
TEAHF [49,50] solution (1 M in THF/dioxane, 1.08 mmol, 1 mL) was added at room temperature to a
solution of 15 (0.254 g, 0.36 mmol) in THF (1 mL) and kept overnight at room temperature. The reaction
4
was quenched with NH Cl solution and extracted with ethyl acetate (3 × 15 mL). The combined extracts
4
were dried over MgSO , concentrated under vacuum. The residue was co-evaporated with anhydrous
pyridine (4 × 5 mL) and redissolved in pyridine (2 mL). 4,4′-Dimethoxytrityl chloride (1.2 eq) was
added in portions at room temperature. The mixture was stirred for 3 h at room temperature, and the
reaction was quenched with methanol (1 mL) and saturated aqueous NaHCO
3
(10 mL). The mixture was
, concentrated under
extracted with DCM. The combined organic extracts were dried over MgSO
4
vacuum and the resulting orange oil was purified by chromatography with 2%–3% MeOH in DCM
1
(
(
with 0.2% of pyridine) as the eluent to give 16 (0.231 g, 72% yield ) as a white solid. H-NMR
CDCl
.74 (s, 6H, 2OCH
m, 8H, spermine). MS (ESI) m/z: calcd for C40
3
): δ (ppm) 7.19–7.36 (m, 9H, DMT), 6.78 (d, 4H, J = 8.6 Hz, DMT), 4.21 (dt, 1H, COCHOH),
3
3
), 3.0–3.44 (m, 14H, CH
2
OH, spermine), 2.18–2.19 (m, 2H, OCHCH
2
), 1.56–1.88
+
(
H
46
F
9
N
4
O
8
[M + K] 933.2887, found 933.279.
2-(4,4′-Dimethoxytrityl)-4-(hydroxymethyl)-N-((2,2,2-trifluoroacet-1-yl)-4,9,13-triazatridecane)-
butyramide]phosphoramidite (17). 17 was synthesized according to procedure described for 11. (0.155 g,
1
5
6
2
5% yield). H-NMR (CDCl
3
): δ (ppm) 6.76–7.4 (m, 13H, DMT), 4.22 (dt, 1H, COCHOH), 3.74 (s,
OH, CONHCH (spermine), CH OP), 2.56–2.62 (m, 2H, 2CH, i-Pr),
CN), 1.57–1.84 (m, 8H, spermine), 1.1–1.2 (m, 12H, 4CH , i-Pr).
H, 2OCH
3
), 3.12–3.55 (m, 16H, CH
2
2
2
.24–2.42 (m, 4H, OCHCH
2
, CH
2
3
31P-NMR (C
H
6
): δ (ppm) 151.73; 151.59; 148.25; 147.83. MS (ESI) m/z: calcd for C50
H
F
N
O P
6
64
9
6
9
+
+
[M + K] 1133.3966, found 1133.303; calcd for C50
64 9 6 9
H F N NaO P [M + Na] 1117.4226, found 1117.45.
3
.3. Oligonucleotide Preparation
The DNA dodecamers (5′-CTC AAG CAA GCT-3′, 5′-AGC TTG CTT GAG-3′, 5′-CTC ACA TGC
GCG-3′, 5′-CGC GCA TGT GAG-3′) were synthesized using DNA synthesizer Gene Assembler Plus
from Pharmacia-LKB (Uppsala, Sweden) or K & A Laborgerate GbR DNA/RNA (Frankfurt am Main,

Molecules 2015, 20
12665
Germany), using standard phosphoramidite chemistry. The non-nucleosidic analogues (6, 11, 11a, 17)
were inserted at positions marked with an X as listed in the Table 1. For the modified phosphoramidites,
two-fold excess of phosphoramidites (in comparison to the standard protocol) and a prolonged coupling
step of 10 min were used. The oligomers were cleaved from the CPG-support with 32% aqueous ammonia
(
room temperature, 1 h). The deprotection under standard conditions using concentrated aqueous
ammonia at 55 °C overnight allowed for removal of all protecting groups, including trifluoroacetyls [3,38].
The oligomers were purified by the TLC on Merck 60 F254 TLC plates with n-propanol/aqueous
ammonia/water solution (55:35:10, by vol.) as an eluent. The product band (least mobile) was cut out,
eluted with water and desalted with Waters Sep-pak C-18 cartridges. First, the solution containing the
oligonucleotide was loaded onto the cartridge and the column was flushed with 10 mM ammonium
acetate (10 mL). In the next step, the oligonucleotides were eluted by flushing the cartridge with 5 mL
of 30% acetonitrile/water solution. The fraction with the product was evaporated to dryness and the
purity of oligonucleotides was monitored using HPLC and confirmed (by MALDI-TOF spectrometry
Autoflex, Bruker). Oligonucleotides were also purified in a subsequent reverse phase HPLC step
(UFLC system with LC-20AD pump; C(18)2 100 Å column (15 cm × 4.6 mm); starting from 0.01 M
triethylammonium acetate (pH 7.0) up to CH CN:CH COOEt N (40%/60%). Identity was confirmed
3
3
3
by mass spectroscopy on MALDI-TOF (Autoflex, Bruker Daltonik GmbH, Bremen, Germany). The
isolated yields of modified oligonucleotides were at the range of 23%–57%: 26%–57% and 23%–26%
for ON3–ON6 and ON7–ON9 respectively.
3
.4. Thermodynamic Analysis
UV melting profiles of the DNA duplexes were obtained in a buffer containing 100 mM sodium
−3
−6
chloride, 20 mM sodium cacodylate, 0.5 mM Na EDTA, pH 7.0. Duplexes were used in the 10 –10 M
2
concentration range. Single strand concentrations were calculated from absorbance above 80 °C with
single strand extinction coefficients approximated by the nearest-neighbor model [51]. The temperature
range, in a heating-cooling cycle, was 0–90 °C with a temperature gradient of 1 °C/min. Thermal-induced
transitions of each mixture were monitored at 260 nm with a Beckman DU 650 spectrophotometer
with a temperature controller. The thermodynamic parameters were determined from fits of data acc.
to a two-state model with the MeltWin 3.5 software [52].
4
. Conclusions
Nucleic acids that carry polycationic modifications have many therapeutic and biotechnological
advantages. Our studies corroborate that non-nucleosidic analogues of polyaminooligonucleosides maintain
affinity and easily form duplexes with complementary strands. In some cases, this may increase the
stability of modified complexes of nucleic acids. These new properties based on the rationale of charge
masking do not change the scheme of the secondary structure of nucleic acids. Thus, they can ease
transferring of nucleic acids past cellular barriers.
Supplementary Materials
Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/20/07/12652/s1.

Molecules 2015, 20
12666
Acknowledgments
This work was supported by the Polish Ministry of Science and Higher Education.
Author Contributions
W.T.M. designed research and supervised the project, J.B. performed all experiments. Both authors
analyzed the data, wrote the manuscript and approved the final version of the manuscript.
Conflicts of Interest
The authors declare no conflict of interest.
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