Synthesis of novel cationic spermine-conjugated phosphotriester oligonucleotide for improvement of cell membrane permeability

Article abstract of DOI:10.1016/j.bmcl.2015.06.071

A spermine-conjugated ethyl phosphotriester oligonucleotide was obtained by solid-phase synthesis based on phosphoramidite chemistry. The ethyl phosphotriester linkage was robust to exonuclease digestion and stable in fetal bovine serum. Cell membrane permeability of the spermine-conjugated ethyl phosphotriester oligonucleotide was studied by fluorescence experiments. The effective cell penetrating potency of the spermine-conjugated ethyl phosphotriester oligonucleotide was determined by confocal laser scanning microscopy and measurement of intracellular fluorescence intensity.

Full text of DOI:10.1016/j.bmcl.2015.06.071

Bioorganic & Medicinal Chemistry Letters 25 (2015) 3610–3615
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis of novel cationic spermine-conjugated phosphotriester
oligonucleotide for improvement of cell membrane permeability
⇑
Junsuke Hayashi, Tomoko Hamada, Ikumi Sasaki, Osamu Nakagawa , Shun-ichi Wada, Hidehito Urata
Osaka University of Pharmaceutical Sciences, 4-20-1 Nasahara, Takatsuki, Osaka 569-1094, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
A spermine-conjugated ethyl phosphotriester oligonucleotide was obtained by solid-phase synthesis
based on phosphoramidite chemistry. The ethyl phosphotriester linkage was robust to exonuclease diges-
tion and stable in fetal bovine serum. Cell membrane permeability of the spermine-conjugated ethyl
phosphotriester oligonucleotide was studied by fluorescence experiments. The effective cell penetrating
potency of the spermine-conjugated ethyl phosphotriester oligonucleotide was determined by confocal
laser scanning microscopy and measurement of intracellular fluorescence intensity.
Ó 2015 Elsevier Ltd. All rights reserved.
Received 24 April 2015
Revised 15 June 2015
Accepted 18 June 2015
Available online 27 June 2015
Keywords:
Cationic oligonucleotide
Phosphotriester
Spermine
Cellular uptake
Oligonucleotide (ON) therapy based on antisense effects is an
important therapeutic tool for various diseases.1 Antisense ON
binding to target mRNA results in the inhibition of specific RNA
processing or translation. However, the use of ONs as a therapeutic
tool is limited as ONs are rapidly digested by nucleases in vivo and
the negative charge of the sugar-phosphate backbone is incompat-
ible with cell membrane permeability. Phosphotriester (PTE) ON is
one of the phosphate-backbone-modified ONs developed in the
1970s.2,3 PTE ON is a promising candidate for effective antisense
ON as the phosphate modification increases resistance to nuclease
digestion in cell culture4 and improves cell membrane permeabil-
ity due to the absence of negative charges on the phosphate moi-
eties.5 More recently, bioreversible PTE linkage-contained RNA
were developed as effective RNAi prodrugs.6 However, neutral
ONs, such as methylphosphonate and PTE ONs, exhibit poor solu-
bility in water due to the modification.7 This indicates that the con-
ventional purification by reversed-phase and anion-exchange
HPLC is often difficult in PTE ON synthesis.
higher affinity to complementary strands than natural PDE ON
due to the decrease of electrostatic repulsion,12,13 and also exhibits
effective cell penetrating potency by electrostatically anchoring to
the cell surface.14 However, a large polycation moiety is required to
neutralize the large number of negative charges on a long ON and
the large polycation moiety exhibits cytotoxicity in living cells.15
In this study, we designed spermine-conjugated PTE ON (Fig. 1)
as a novel cationic ON. The conjugation of PTE ON, which has no
negative charge, with spermine may resolve issues surrounding
both PTE ON and the polycation moiety, as the positive charges
on the spermine moiety may increase the solubility of PTE in
water, and a large polycation moiety is unnecessary for PTE ON
to be cationic. We present herein the solid-phase syntheses of
spermine-conjugated PTE ONs and their effective cell membrane
permeability.
Generally, PTE linkages are unstable under harsh basic condi-
tions, such as concentrated aqueous ammonia employed to remove
the N-acyl protective groups.16 In order to confirm the stability of
PTE ON under deprotection conditions, model PTE thymidine
dimers (5a, b) were synthesized. As shown in Scheme 1, methyl
(3a) and ethyl (3b) phosphoramidite units were synthesized from
50-O-protected thymidine (1) in two steps in 57% and 56% yields,
respectively. Those amidite units were subjected to the reaction
with 30-O-protected thymidine in the presence of 1H-tetrazole as
an activator, followed by oxidation with 2-butanone peroxide in
toluene solution and the removal of 30,50-O-protections with 80%
AcOH, to afford methyl (5a) and ethyl (5b) PTE dimers in 40%
and 32% overall yields in six steps from thymidine, respectively.
Cationic ONs have been studied in an effort to improve the cell
membrane permeability of ONs.8,9 Cationic ONs, which are conju-
gates of natural phosphodiester (PDE) ON with polycation moi-
eties, such as peptides10 and polyamines,11 have been reported.
Recently, it was reported that spermine-conjugated PDE ON has
⇑
Corresponding author. Tel./fax: +81 72 690 1089.
E-mail address: urata@gly.oups.ac.jp (H. Urata).
Present address: Graduate School of Pharmaceutical Sciences, Osaka University,
1-6 Yamadaoka, Suita, Osaka 565-0871, Japan.
http://dx.doi.org/10.1016/j.bmcl.2015.06.071
0960-894X/Ó 2015 Elsevier Ltd. All rights reserved.

J. Hayashi et al. / Bioorg. Med. Chem. Lett. 25 (2015) 3610–3615
3611
Figure 1. Spermine-conjugated PTE T10
.
Scheme 1. Synthesis of phosphotriester (PTE) dimer 5. (i) 4,40-dimethoxytrityl chloride (DMTrCl), pyridine, rt, 2.5 h; (ii) [(i-Pr)2N]2PCl, N,N-diisopropylethylamine, CH2Cl2, rt,
2 h; (iii) ROH, 1H-tetrazole, CH2Cl2, rt, 2 h; (iv) 30-O-DMTr thymidine, 1H-tetrazole, CH2Cl2, rt, 3 h; (v) 6.7% 2-butanone peroxide in toluene, rt, 5 min; (vi) 80% AcOH aq, rt,
1.5 h.
The structures of dimers were characterized by 31P NMR and mass
spectrometry.17
incubated under enzymatic digestive conditions.20 The reactions
were analyzed by reversed-phase HPLC and the peak areas of
the intact dimers in the chromatograms were calculated. After
incubation for 180 min, natural parent PDE dimer was rapidly
hydrolyzed by SVPDE, whereas ethyl PTE dimer was extremely
stable (Fig. 3). Similar results were obtained in 10% FBS. The
results suggest that the PTE linkage is sufficiently stable
in vitro and in vivo.
Next, we synthesized spermine phosphoramidite unit 10
according to literature procedure21 with slight modifications,
enabling shorter step synthesis (Scheme 2). The amino groups of
spermine tetrahydrochloride were protected by trifluoroacetyl
(TFA) groups, and subsequently the protected terminal amide
groups were reacted with tert-butyl(4-iodobutoxy)dimethylsilane
(TBDMSO(CH2)4I) in the presence of sodium hydride (NaH) to
afford 7. Removal of the TBDMS group with tetrabutylammonium
fluoride (TBAF) afforded compound 8. One terminal hydroxyl
group of 8 was protected by a 4,40-dimethoxytrityl (DMTr) group
and finally, the other hydroxyl group was phosphitylated to afford
spermine phosphoramidite unit 10. The overall yield of 10
obtained from spermine tetrahydrochloride was 11%.
The stability of PTE dimers 5a and 5b under standard (conc. NH3
aq at 55 °C for 8 h) and mild (conc. NH3 aq at rt for 2 h) deprotec-
tion conditions was studied. The reactions were analyzed by
reversed-phase HPLC. Diastereomeric peaks were found in the
HPLC chromatograms of untreated dimers 5a and 5b
(Fig. 2A and D). After treatment with conc. NH3 aq at 55 °C for
8 h, methyl PTE dimer 5a was completely degraded into PDE dimer
d(TpT), whereas ethyl PTE dimer 5b was partially degraded
(Fig. 2B and E). On the other hand, after treatment with conc.
NH3 aq at rt for 2 h, ethyl PTE 5b was stable, whereas methyl
PTE 5a was almost completely degraded (Fig. 2C and F). The results
indicate that labile protective groups, such as phenoxyacetyl (Pac)
for dA, isopropylphenoxyacetyl (i-PrPac) for dG, and acetyl (Ac) for
dC groups, are suitable for the synthesis of ethyl PTE ON.
To confirm the stability of the ethyl PTE dimer against exonu-
clease (snake venom phosphodiesterase, SVPDE) and in serum
(fetal bovine serum, FBS), ethyl PTE d(CpT), which is synthesized
according to the above procedure using allyoxycarbonyl (Alloc)
protection for dC,18,19 and parent PDE d(CpT) dimers were

3612
J. Hayashi et al. / Bioorg. Med. Chem. Lett. 25 (2015) 3610–3615
Figure 2. HPLC analysis of the degradation of methyl (5a) and ethyl (5b) PTE dimers under alkaline conditions. Dimers were treated under deprotection conditions. (A and D)
untreated; (B and E) 28% NH3 aq, 55 °C, 8 h; (C and F) 28% NH3 aq, rt, 2 h. The HPLC analysis was conducted on a Shimadzu LC-6A system with a column of -Bondasphere
l
5C18 column (3.9 ꢀ 150 mM, Waters). Elution was performed with a linear gradient of acetonitrile 5–25% for 20 min in 50 mM triethylammonium acetate, pH 7.0.
Figure 3. Comparison of the stability of d(CpT) ethyl PTE (d) and PDE (N) dimers. The vertical axis of the graph indicates the ratio of intact dimers in the reactions. (A) Against
SVPDE, (B) in 10% FBS.
To study the cell membrane permeability of spermine-conju-
gated PTE thymidine 10mer (T10), fluorescent 30-TAMRA-labeled
50-O-spermine-conjugated PTE T10 14 was synthesized. At first,
30-TAMRA-CPG was coupled with thymidine ethyl phospho-
ramidite unit 3b to form a PTE linkage between PTE T10 and
TAMRA. However, the PTE linkage at the 30-end was cleaved
by nucleophilic attack of the hydroxyl group of the 30-TAMRA
linker under alkaline deprotection conditions (Scheme 3). To
cyanoethyl phosphoramidite unit was used in the first coupling
step with 30-TAMRA-CPG, and this was followed by the coupling
of thymidine ethyl phosphoramidite unit 3b with CPG
(Scheme 4). The coupling reaction was conducted with 5-
ethylthio-1H-tetrazole (ETT) as the activator, and oxidation of
trivalent phosphorus was carried out with 0.02 M iodine solu-
tion. Finally, the 50-hydroxyl group of PTE T10 was coupled with
spermine phosphoramidite unit 10 to obtain the spermine-con-
jugated ON. The deprotection and cleavage from the support
avoid this reaction,
a
commercially available thymidine

J. Hayashi et al. / Bioorg. Med. Chem. Lett. 25 (2015) 3610–3615
3613
Scheme 2. Synthesis of spermine phosphoramidite unit 10. (i) TFA2O, pyridine/CH2Cl2, rt, 22 h; (ii) TBDMSO(CH2)4I, NaH, DMF, rt, 2 h; (iii) tetrabutylammonium fluoride,
THF, rt, 1 h; (iv) DMTrCl, pyridine/CH2Cl2, rt, 2 h; (v) CNEtOP[N(i-Pr)2]2, diisopropylammonium tetrazolide, CH2Cl2, rt, 2 h.
Scheme 3. Cleavage of PTE T10-30-TAMRA linker under alkaline conditions.
was conducted with conc. NH3 aq at room temperature for 2 h.
Reversed-phase HPLC analysis and purification of 30-TAMRA-la-
beled 50-O-spermine-conjugated PTE T10 14 were performed in
50 mM triethylammonium acetate buffer (pH 4.3) (Scheme 4).
As the control, 30-TAMRA-labeled 50-O-spermine-unconjugated
PTE T10 13, 30-TAMRA-labeled 50-O-spermine-conjugated PDE
T10 12, and 30-TAMRA-labeled 50-O-spermine-unconjugated PDE
T10 11 were also synthesized (Table 1). After purification, the
structures were characterized by MALDI-TOF mass spectrometry.
As expected, spermine-conjugated ON 14 exhibited much
improved solubility in water compared to spermine-
unconjugated ON 13. For this reason, small amount of DMSO
(ca. 10%) was added to the stock solution of ON 13.
ONs 11–14 were dissolved in aqueous buffer (10 mM Tris–
HCl, 1 mM EDTA, pH 8) and their cell membrane permeability
was studied by confocal laser scanning microscopy (CLSM) using
human alveolar basal epithelial lung adenocarcinoma (A549)
cells. The cells were incubated with fluorescently labeled ONs
11–14 at the concentration of 10 lM for 8 h at 37 °C in serum-
free medium (Opti-MEM) without any transfection reagent.
After incubation for 8 h, strong fluorescence was observed when
the cells were incubated with 50-O-spermine-conjugated PTE T10

3614
J. Hayashi et al. / Bioorg. Med. Chem. Lett. 25 (2015) 3610–3615
Scheme 4. Synthesis of 30-TAMRA-labeled 50-O-spermine-conjugated PTE T10
.
Table 1
subsequently, the fluorescence intensities were measured by a
spectrofluorometer (Ex at 543 nm and Em at 580 nm). As shown
in Figure 5, PTE-type ONs 13 and 14 demonstrated 16 and 6.1
times stronger fluorescence intensities than PDE-type ONs 11
and 12, respectively. Furthermore, spermine-conjugated ONs 12
and 14 showed 5.5 and 2.1 times stronger fluorescence intensi-
ties than spermine-unconjugated ONs 11 and 13, respectively.
The results indicate that the PTE ONs exhibit higher cell mem-
brane permeability than the PDE ONs, and the spermine conju-
gation increases the cell membrane permeability of PDE and
PTE ONs.
Synthesized fluorescent 30-TAMRA-labeled T10 11–14
ON
PDE or PTE
Spermine
MALDI-TOF massa
Calcd. (M+H)+
Found
11
12
13
14
PDE
PDE
PTE
PTE
Unconjugated
Conjugated
Unconjugated
Conjugated
3604.7
3857.0
4013.1
4265.3
3605.3
3857.4
4013.6
4266.0
a
Matrix: A mixture of 3-hydroxypicolinic acid (3-HPA)/diammonium hydrogen
citrate = 9/1.
In summary, we synthesized spermine-conjugated ethyl PTE T10
14 using the solid-phase phosphoramidite method. The PTE link-
age exhibited sufficient stability against enzymatic digestion
in vitro and the spermine-conjugated PTE ON displayed excellent
cell membrane permeability without having to use any transfec-
tion reagent. Studies of the antisense effect of spermine-conju-
gated PTE ON are ongoing in our laboratory.
14, and no significant fluorescence was observed when the cells
were incubated with control ONs 11–13 (Fig. 4). Next, to esti-
mate the intracellular fluorescence intensities of the ONs, fluo-
rescence analyses of the cell lysates were conducted. After
A549 cells were incubated with the ONs for 8 h, the cells were
washed with PBS and treated with lysis buffer, and

J. Hayashi et al. / Bioorg. Med. Chem. Lett. 25 (2015) 3610–3615
3615
Figure 4. CLSM images of A549 cells. Cells were incubated with 30-TAMRA-labeled ON at the concentration of 10
spermine-conjugated PDE ON 12; (C) PTE ON 13; (D) spermine-conjugated PTE ON 14.
lM in Opti-MEM for 8 h at 37 °C. (A) PDE ON 11; (B)
Figure 5. Comparison of intracellular fluorescence of ONs 11–14. A549 cells were incubated with each 10
fluorescence intensities of cell lysates were analyzed by a spectrofluorometer (n = 4).
lM ON in Opti-MEM for 8 h at 37 °C. After the washing steps,
11. Polushin, N. N. Nucleic Acids Res. 2000, 28, 3125.
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Acknowledgments
We thank the Cell Resource Center for Biomedical Research
(Institute of Development, Aging and Cancer, Tohoku University)
for providing A549 cells.
13. Pons, B.; Kotera, M.; Zuber, G.; Behr, J.-P. ChemBioChem 2006, 7, 1173.
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References and notes
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for C21H30N4O12P [M+H]+ 561.1597 found 561.1601; 5b: 31P NMR (121 MHz,
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575.1753 found 575.1749.
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19. The structure of ethyl PTE d(CpT) was confirmed by 31P NMR and mass
spectrometry. 31P NMR (121 MHz, CD3OD) d: ꢁ0.83, ꢁ1.03. HR-MS (FAB): calcd
for C21H31N5O11P [M+H]+ 560.1757 found 560.1761.
20. Dimers (5 nmol) were dissolved in 10 mM MgCl2, 50 mM Tris–HCl, pH 8.0
(395
(40
Aliquots of the reactions (40
and 180 min and the samples were heated at 90 °C for 2 min (for SVPDE) or
mixed with formamide (20 l) (for FBS) to terminate the reactions. The
l
l
l) for SVPDE or water (360
l) was added to those solutions. The solutions were incubated at 37 °C.
l each) were removed at 0, 1, 5, 10, 20, 40, 90,
ll) for FBS. SVPDE (8 lg/ml) (5 ll) or FBS
l
l
samples were analyzed by reversed-phase HPLC.
21. Voirin, E.; Behr, J.-P.; Kotera, M. Nat. Protocols 2007, 2, 1360.
10. Fraley, A. W.; Pons, B.; Dalkara, D.; Nullans, G.; Behr, J.-P.; Zuber, G. J. Am. Chem.
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