Synthesis and cell transfection properties of cationic uracil-morpholino tetramer

Article abstract of DOI:10.1016/j.tetlet.2013.12.087

Synthesis and cell transfection properties of guanidinium-functionalized uracil morpholino tetramer have been reported for the first time. Due to the basic nature of guanidinium groups they remain protonated under physiological conditions. Such cationic tetramer exhibits efficient cellular uptake properties as visualized by microscopy imaging using fluorescent dye BODIPY. 7′-End of this morpholino tetramer was functionalized with an azide group for conjugation with various types of biomolecules or drugs for cellular delivery.

Full text of DOI:10.1016/j.tetlet.2013.12.087

Tetrahedron Letters 55 (2014) 1072–1076
Contents lists available at ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Synthesis and cell transfection properties of cationic
uracil-morpholino tetramer
⇑
Sibasish Paul, Sankha Pattanayak, Surajit Sinha
Department of Organic Chemistry, Indian Association for the Cultivation of Science, Jadavpur, Kolkata 700 032, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Synthesis and cell transfection properties of guanidinium-functionalized uracil morpholino tetramer
have been reported for the first time. Due to the basic nature of guanidinium groups they remain proton-
ated under physiological conditions. Such cationic tetramer exhibits efficient cellular uptake properties
as visualized by microscopy imaging using fluorescent dye BODIPY. 70-End of this morpholino tetramer
was functionalized with an azide group for conjugation with various types of biomolecules or drugs
for cellular delivery.
Received 22 October 2013
Revised 20 December 2013
Accepted 24 December 2013
Available online 2 January 2014
Keywords:
Uracil morpholino
Nucleosides
Ó 2013 Elsevier Ltd. All rights reserved.
Cationic morpholino
Cell transfection
Fluorescent imaging
Phosphorodiamidate morpholino oligos (PMO)1 are synthetic
oligonucleotides having two major structural differences; the neg-
atively charged phosphorodiester internucleoside linkage in DNA
has been replaced by neutral phosphorodiamidate linkage and
the five-membered ring of deoxyribose in DNA is replaced by the
six-membered morpholine ring (Fig. 1). PMOs are routinely used
for gene silencing when they are designed for targeting 50-UTR of
mRNA.2 The extraordinary stability, lack of net charge on the inter-
linkage backbone, and total resistance to metabolic degradation
have made this class of molecules worthy of consideration as
potential therapeutics. The growing clinical data indicate that
phosphorodiamidate morpholinos are safe during Phase I/II clinical
trials and appear to have a favorable pharmacological profile.3
Incorporation of T- or U-morpholino unit(s) into DNA has also been
reported to obtain better efficacy of these oligomers as antisense
compounds.4–6 Similarly, chimeric DNAs with phosphoramidate
morpholino modification show better resistance toward the nucle-
ase activity.7 Due to the application of morpholino triphosphates as
chain terminating reagents in DNA sequencing,8 synthesis of these
triphosphates has been reported as nucleoside analogues.4 Apart
from their antisense activity, these oligomers have also found their
growing applications in nanotechnology,9 in surface hybridiza-
tion10,11 as neutral DNA analogues. Though PMOs are stable under
enzymatic degradation, however, their cellular transfection prop-
erty is very poor which precludes their therapeutic application
and in tissue culture as well. The typical length for a PMO to
achieve effective antisense activity is between 18 and 25 bases.2
However, at this size, passive diffusion through cell membranes
is slow. Thus, guanidinium-rich cell penetrating peptides (CPP) or
cationic dendrimers are covalently conjugated to PMOs and the
conjugated PPMOs can penetrate a wide variety of cell types.12
However, the stability of CPPs in the presence of peptidases12b
sometimes becomes a problem and can generate toxicity12c and
pose risks of immunogenicity on long-term treatment.12d Scientists
are involved to develop different carriers for delivery of this class of
antisense molecules. In 2005, a group from Japan reported that
nucleobases with guanidinium modification exhibit enhanced cel-
lular uptake.13a Recently, we have developed cell penetrating mor-
pholinos by replacing the phosphorodiamidate interlinkages by
guanidinium linkages.13b Since the guanidinium groups are highly
basic (pKa ꢀ12.5) in nature and they can provide directionality via
hydrogen bonding due to their planar structure, introduction of
guanidinium groups at suitable positions of oligonucleotide can
improve their pharmacodynamic and pharmacokinetic properties.
Moreover the higher pKa introduces a positive charge that is main-
tained over a wide range of pH. Keeping these studies in mind, we
herein report the synthesis and cell transfection properties of
guanidinylated uracil morpholino tetramer (Fig. 1). Fluorescent
dye was attached with a linker at the 40-end of tetramer for imag-
ing purpose. An azide group at the 70-end of the tetramer was
introduced as this can be used for further conjugation with biomol-
ecules or drugs for delivery inside the cells through click reaction.
Solution phase synthesis of such base modified morpholino oligo-
mers and their cell penetration properties have been revealed for
the first time.
⇑
Corresponding author. Tel.: +91 33 2473 4971; fax: +91 33 2473 2805.
E-mail address: ocss5@iacs.res.in (S. Sinha).
0040-4039/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tetlet.2013.12.087

S. Paul et al. / Tetrahedron Letters 55 (2014) 1072–1076
1073
HN
O
N
O
N
O
N
NH2
HN
N
H
HN
HN
O
O
O
O
O
NH
NH2
O
N
O
O
O
O
N
O
O
N
N
H
O
P
HN
N
P
O
N
P
O
HN
HN
O
O
Me2N
O
Me2N
O
O
O
O
O
O
O
O
N
N
O
a
b
c
Figure 1. Chemical structure of (a) DNA; (b) Morpholino ODN and (c) Guanidinium substituted morpholino oligo (GMO).
For the synthesis of the key starting material chlorophosph-
oramidate 6, we began from naturally occurring ribonucleoside
uridine (Scheme 1). Protection using TBDPSCl delivered the mono-
protected nucleoside 1. Oxidative cleavage of C–C bond was carried
out using NaIO4 in methanol, followed by formation of morpholine
nucleus using ammonium biborate in the same pot. Reduction
using NaCNBH3 gave the desired morpholino amine 2.14 N-Protec-
tion was carried out using trityl chloride in dry DMF. Removal of
the silyl group was performed using tetrabutylammonium fluoride
in THF to obtain the corresponding alcohol 3. Iodination was
carried out using ICl in MeOH and potassium carbonate as a base
to obtain compound 4 in 83% yield.15 Sonogashira reaction was
achieved following our lab protocol to obtain compound 5 in good
yield.15 Activated chlorophosphoramidate (6) was synthesized in
47% yield using LiHMDS as base.16
morpholino timer 9. Similar iterative reaction sequences provided
morpholino tetramer 10 (Scheme 2).
Morpholino tetramer 10 was then functionalized with linker 11
to obtain compound 12. Compound 11 was synthesized from ami-
nocaproic acid following the standard literature protocol.17 Det-
ritylation, followed by conjugation with fluorescent dye BODIPY
13 gave compound 14. To incorporate guanidinium groups through
alkyne spacer at the C-5 position of morpholino oligomers, we then
deprotected all trifluoroacetamide groups from 14. Deprotection
reaction was carried out using 10% MeOH in ammonia to obtain
the free terminal amines. Guanidinylation reaction using
pyrazole-1-carboxamidine hydrochloride following Wender’s pro-
cedure failed to produce our desired product.18 The tri-urethane
protected guanidinium reagents were also unsuccessful to provide
the guanidinium morpholino tetramer.19 However, using O-meth-
ylisourea hydrochloride in 30% ammonia solution at 55 °C gave
the cationic morpholino tetramer 15 (Scheme 3).20,21 Compound
15 was then purified by preparative TLC and obtained in 23% yield
after two steps from compound 14.
The 70-azido morpholino monomer unit 7, was synthesized
from the corresponding N-tritylated monomer 3. Mesylation,
followed by treatment with NaN3 and NH4Cl gave the azido mono-
mer 7 in 70% yield.13b Detritylation of compound 7 was carried out
using acetic acid in trifluoroethanol, followed by coupling with
activated chlorophosporamidate 6 that gave morpholino dimer 8.
Detritylation of dimer 8 followed by coupling with 6 provided
Since, cellular uptake efficiency is known to be highly depen-
dent on cell type; we extended our studies from Chinese hamster
ovary (CHO K1) to prostate adenocarcinoma (PC-3) cells. Cells in
O
O
O
N
NH
NH
NH
N
O
HO
N
O
O
TBDPSO
i
ii, iii
O
TBDPSO
iv, v
O
O
N
H
OH OH
OH OH
2
1
3
F3COCHN
F3COCHN
O
O
O
N
O
N
I
NH
NH
NH
N
O
vii
Cl
P
O
vi
O
viii
HO
O
HO
N
O
O
O
N
Ph
Ph
N
N
Ph
Ph
Ph
Ph
Ph
Ph
Ph
4
5
6
Scheme 1. Synthesis of uracil-modified morpholino monomers. Reagents and conditions: (i) TBDPSCI, imidazole, DMF; (ii) NaIO4, (NH4)B4O7, MeOH; (iii) NaCNBH3, 4 Å MS,
AcOH, MeOH, 55% after three steps; (iv) ClCPh3, Et3N, DMF, 83%; (v) TBAF, THF, 88%; (vi) ICI, K2CO3, MeOH, 83%; (vii) 2,2,2-trifluro-N-(prop-2-yn-1-yl)acetamide, Pd(PPh3)4,
CuI, Et3N, DMF, 86%; (viii) LiHMDS, Cl2NMe2(O)P, THF, 47%.

1074
S. Paul et al. / Tetrahedron Letters 55 (2014) 1072–1076
O
O
N
O
N
HN
HN
HN
O
N
O
O
ii
iii, iv
i
MsO
N3
HO
O
O
O
N
N
N
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
7
3
O
N
HN
O
O
HN
NHCOCF3
N3
O
O
O
N
NHCOCF3
iii, iv
N
P
O
N3
HN
O
O
N
Me2N
O
N
P
O
O
N
HN
NHCOCF3
Me2N
O
O
O
O
N
O
HN
Me2N
P
O
O
O
N
Ph
Ph
N
Ph
O
N
8
9
Ph
Ph
O
N
Ph
HN
O
NHCOCF3
N3
O
O
N
N
P
O
HN
Me2N
O
O
NHCOCF3
v, vi
O
O
N
HN
Me2N
P
O
O
O
N
NHCOCF3
O
O
N
10
N
HN
Me2N
P
O
O
O
O
N
Ph
Ph
Ph
Scheme 2. Solution phase synthesis of morpholino tetramers. Reagents and conditions: (i) MsCl, Pyridine; (ii) NaN3, NH4Cl, DMF, 70% after two steps; (iii) 3%, AcOH,
CF3CH2OH; (iv) 6, DIPEA, CH3CN, rt; (v) 4%, AcOH, CF3CH2OH; (vi) 6, DIPEA, DMF, rt.
culture were treated with BODIPY-tagged cationic PMO 15 to give a
final concentration of 3.0 M in opti-MEM media, and monitored
In summary, we have synthesized guanidinium-substituted
uracil morpholino monomer and cationic uracil morpholino tetra-
mer for the first time. Such cationic oligomer has cell transfection
property, confirmed by fluorescence imaging in CHO and PC-3
cells. The modified morpholino oligos can be used as delivery
agents for biomolecules, drugs etc. through conjugation. Addition-
ally, incorporation of such modified morpholino units into oligonu-
cleotide backbone can solve the long standing problem of cellular
transfection properties of antisense compounds.
l
its fate under fluorescence microscope. After 18 h of incubation
at standard conditions, fluorescence was observed mainly within
the cytoplasm and to a very low amount in the nucleus. The cyto-
plasmic fluorescence appeared to be distributed throughout the
cytoplasm in a diffuse manner and was internalized in a dose-
dependent manner as can be seen from Figure 2a and c. PC-3 cells
were fixed with formaldehyde before analysis and live cell images
were taken in case of CHO cells.22 Role of guanidinium groups can
be understood as N-acetylated compound 14 displayed very poor
cellular uptake under similar cell-culture conditions (Fig. 2f). The
positive charge of guanidinium groups is presumed to form an ini-
tial complex with the cell surface followed by cellular uptake
through energy dependent endocytosis process.23
Acknowledgements
S.S. thanks the Department of Biotechnology, Government of In-
dia, for financial support by a Grant no. [BT/PR13426/BRB/10/761/
2009]. S.P. and S.P. are grateful to CSIR for their fellowship.

S. Paul et al. / Tetrahedron Letters 55 (2014) 1072–1076
1075
O
HN
O
N
NHCOCF3
N3
O
O
N
N
P
O
n
iii, iv
HN
i, ii
Me2N
O
10
O
NHCOCF3
O
O
O
N
N
N
B
N
N
O
NHCPh3
HN
F
F
O
5
Me2N
P
O
O
O
O
O
O
O
13
11
N
O
O
12
NHCPh3
N
n= 2
N
O
5
H
O
N
O
N
HN
NH
NH2
HN
O
HN
O
NHCOCF3
N3
O
O
N
N3
O
O
N
P
O
n
HN
N
P
O
n
NH
NH2
HN
Me2N
O
Me2N
O
O
HN
O
N
NHCOCF3
O
O
O
O
v, vi
N
N
HN
HN
Me2N
P
O
O
O
Me2N
P
O
O
O
N
N
O
O
N
N
F
N
N
N
F
B
N
B
O
O
F
O
F
O
15
n= 2,
14
n= 2,
NH
NH
Scheme 3. Synthesis of cationic morpholino tetramer 15. Reagents and conditions: (i) 4%, AcOH, CF3CH2OH; (ii) 11, DIPEA, DMF, rt; (iii) 4%, AcOH, CF3CH2OH; (iv) 13, DIPEA,
DMF, rt; (v) 10%, NH3 in MeOH; (vi) O-mehylisoureahydrochloride, 30% NH3 in water 55 °C.
Figure 2. Visualization of guanidinium-linked PMO 15 uptake and intercellular trafficking by fluorescence microscopy after incubation of 18 h in opti-MEM media at 37 °C.
(a) Image showing fluorescein localization in PC-3 cells at 3.0
M concentration. (d) Fluorescein localization image (40X) in CHO cells and (e) Phase contrast image (40X) of CHO cells. (f) Fluorescein localization of 14 in PC-3 cells at
M concentration.
lM concentration; (b) DAPI nuclear localization of the same cells; (c) Fluorescein localization in PC-3 cells at
2
l
3.0
l

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S. Paul et al. / Tetrahedron Letters 55 (2014) 1072–1076
15. Paul, S.; Nandi, B.; Pattanayak, S.; Sinha, S. Tetrahedron Lett. 2012, 53, 4179.
Supplementary data
16. Shestopalov, I. A.; Sinha, S.; Chen, J. K. Nat. Chem. Biol. 2007, 3, 650.
17. Petrie, C. R.; Adams, A. D.; Stamm, M.; Ness, J. V.; Watanabe, S. M.; Meyer, R. B.,
Jr. Bioconjugate Chem. 1991, 2, 441.
18. Wender, P. A.; Jessop, T. C.; Pattabiraman, K.; Pelkey, E. T.; VanDeusen, C. L. Org.
Lett. 2001, 3, 3229.
19. Feichtinger, K.; Sings, H. L.; Baker, T. J.; Matthews, K.; Goodman, M. J. Org. Chem.
1998, 63, 8432.
20. Deglane, G.; Abes, S.; Michel, T.; Prévot, P.; Vives, E.; Debart, F.; Barvik, I.;
Lebleu, B.; Vasseur, J. J. ChemBioChem 2006, 7, 684.
Supplementary data (detailed experimental for all compounds
including 1H and 13C NMR spectral assignments, and other supple-
mentary information) associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.tetlet.2013.
12.087.
21. Preparation of compound 15: Compound 12 (0.004 g) was dissolved in
trifluoroethanol (0.5 mL) followed by addition of acetic acid (0.02 mL)
dropwise. The reaction mixture was stirred at room temperature for 4 h.
Reaction mixture was concentrated to minimum volume. Coevaporated with
EtOAc (2 ꢁ 2 mL), washed with hexane, and dried in vacuo to obtain crude
morpholino amine. To the crude reaction mixture of amine in DMF (0.5 mL)
was added DIPEA (0.005 mL), followed by addition of 13 (0.001 g). The reaction
mixture was stirred at 20 °C for 3 h. Concentrated in vacuo and purified by
preparative TLC (15% MeOH–CHCl3) to obtain compound 14 in 51% yield.
Compound 14 was dissolved in 10% NH3 in MeOH (1.0 mL) and stirred at room
temperature for 1 h in a sealed vessel. After deprotection, the reaction mixture
was evaporated to dryness. To the crude reaction mixture was added a solution
References and notes
1. Summerton, J.; Weller, D. Antisense Nucleic Acid Drug Dev. 1997, 7, 187.
2. Summerton, J. Biochim. Biophys. Acta 1999, 1489, 141.
3. Crooke, S. T. Antisense Drug Technology: Principles, Strategies, and Applications;
CRC, 2007.
4. Abramova, T. V.; Bakharev, P. A.; Vasilyeva, S. V.; Silnikov, V. N. Tetrahedron Lett.
2004, 45, 4361.
5. Perez-Rentero, S.; Alguacil, J.; Robles, J. Tetrahedron 2009, 65, 1171.
6. Zhang, N.; Tan, C. Y.; Cai, P. Q.; Jiang, Y. Y.; Zhang, P. Z.; Zhao, Y. F. Tetrahedron
Lett. 2008, 49, 3570.
7. Zhang, N.; Tan, C. Y.; Cai, P. Q.; Zhang, P. Z.; Zhao, Y. F.; Jiang, Y. Y. Bioorg. Med.
Chem. 2009, 17, 2441.
of O-methylisourea hydrochloride (0.1 g) in water (100
lL) and ammonia (30%,
125 L). Reaction mixture was heated at 55 °C for overnight. The solvent was
l
8. Marciacq, F.; Sauvaigo, S.; Issartelte, J.-P.; Mouret, J. F. C.; Molko, D. Tetrahedron
Lett. 1999, 40, 4673.
removed and compound 15 was purified by preparative TLC (50% MeOH–
CHCl3).
9. Zhang, G.-J.; Luo, Z. H. H.; Huang, M. J.; Tay, G. K. I.; Lim, E.-J. A. Biosens.
Bioelectron. 2010, 25, 2447.
22. Live cell fluorescence imaging using CHO-K1 cells: CHO-K1 cells were plated at
30,000 cells/well in DMEM containing 10% FBS and 100 lg/mL streptomycin
10. Tercero, N.; Wang, K.; Gong, P.; Levicky, R. J. Am. Chem. Soc. 2009, 131, 4953.
11. Wang, X.; Smirnov, S. ACS Nano 2009, 3, 1004.
and 100 units/mL penicillin in 24-well tissue culture plates and allowed to
attach at 37 °C in a humid 5% CO2 atmosphere for 24 h. After removal of media,
12. (a) Moulton, J. D.; Jiang, S. Molecules 2009, 14, 1304; (b) Youngblood, D. S.;
Hatlevig, S. A.; Hassinger, J. N.; Iversen, P. L.; Moulton, H. M. Bioconjugate Chem.
2007, 18, 50; (c) Wu, R. P.; Youngblood, D. S.; Hassinger, J. N.; Lovejoy, C. E.;
Nelson, M. H.; Iversen, P. L.; Moulton, H. M. Nucleic Acids Res. 2007, 35, 5182;
(d) Lee, S. H.; Castagner, B.; Leroux, J.-C. Eur. J. Pharm. Biopharm. 2013, 85, 5.
13. (a) Ohmichi, T.; Kuwahara, M.; Sasaki, N.; Hasegawa, M.; Nishikata, T.; Sawai,
H.; Sugimoto, N. Angew. Chem., Int. Ed. 2005, 44, 6682; (b) Pattanayak, S.;
Khatra, H.; Saha, S.; Sinha, S. RSC Adv. 2014, 4, 1951.
500
morpholino tetramer 15 in DMSO-H2O (1:1) was added to each well so that
3.0 M final concentration was achieved. Cells were incubated with BODIPY-
GMO for 18 h in standard conditions, and the media was removed. The cells
were washed twice with PBS and replaced with fresh media. The cells were
directly visualized under an Olympus inverted fluorescent microscope with a
40X objective (Fig. 2).
lL of 0.5% FBS in DMEM was added to each well. The BODIPY-conjugated
l
23. Madani, F.; Lindberg, S.; Langel, U.; Futaki, S.; Graslund, A. Biophys. J. 2011,
2011, 414729.
14. Pattanayak, S.; Paul, S.; Nandi, B.; Sinha, S. Nucleosides, Nucleotides Nucleic Acids
2012, 31, 763.