Synthesis of hairpin siRNA using 18β-glycyrrhetinic acid derivative as a loop motif

Article abstract of DOI:10.1016/j.tetlet.2009.03.049

We synthesized a new phosphoramidite building block from 18β-glycyrrhetinic acid. This compound was introduced in the middle of the palindromic oligonucleotides and they formed hairpin structure. This 18β-glycyrrhetinic acid derivative excellently perform

Full text of DOI:10.1016/j.tetlet.2009.03.049

Tetrahedron Letters 50 (2009) 2545–2547
Contents lists available at ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Synthesis of hairpin siRNA using 18b-glycyrrhetinic acid derivative
as a loop motif
*
Eun-Kyoung Bang, Byeang Hyean Kim
Department of Chemistry, BK School of Molecular Science, Pohang University of Science and Technology, Pohang 790-784, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
We synthesized a new phosphoramidite building block from 18b-glycyrrhetinic acid. This compound was
introduced in the middle of the palindromic oligonucleotides and they formed hairpin structure. This
18b-glycyrrhetinic acid derivative excellently performed its role as hairpin loop. These hairpin ODNs
had higher Tm values than the natural one and formed stable hairpin structure without any structural dis-
tortion. We found that stability of hairpin ODNs was changed according to the length of hairpin stem and
modified hairpin ODNs, which had longer than seven base pair stem, were more stable than natural one
based on Tm values. The shRNAs bearing similar modified loop were also synthesized. These modified
shRNAs could suppress their target gene expression without losing their RNAi efficiency.
Ó 2009 Elsevier Ltd. All rights reserved.
Received 24 February 2009
Accepted 10 March 2009
Available online 13 March 2009
Keywords:
18b-Glycyrrhetinic acid
Hairpin ODN
shRNA
RNA interference
Nucleic acid has been an attractive material in this century be-
cause of their highly specific recognition between nucleobases.1
Since the solid phase synthesis based on phosphoramidite chemis-
try by automatic synthesizer has been established, it has been pos-
sible for easy modification of oligonucleotide using fuctional
building blocks.2
RNA interference (RNAi) is known as the most effective gene
knock-down process since A. Fire and C. Mello reported.3 In RNAi,
double-stranded RNA known as small interfering RNA (siRNA) is
used to suppress the expression of targeted genes.4 The short hair-
pin RNA (shRNA) also has ability to silence a target gene through
the similar RNAi mechanism.4c The purpose of shRNA was reducing
unspecific side effect and generating the therapeutic siRNAs.5 To
improve their stability, cellular uptake properties, and target spec-
ificity, modification of these therapeutic RNAs has been widely ex-
plored.6 Although various modified siRNAs have been reported,
modified hairpin siRNA (shRNA) is rare.
tracts of licorice and reported as the compound having anti-inflam-
matory, anti-viral, anti-allergic, and anti-tumor activities.13 The
molecule has similar size with cholane-3,24-diol but more rigid
structure, so it could act as a stable loop in the hairpin structure.
We synthesized the phosphoramidite from 18b-glycyrrhetinic
acid as described in Scheme 1. 18b-glycyrrhetinic acid was reduced
by lithium aluminum hydride for 4 h to give 1 in 95% yield.14 After
hydrogenolysis of two diastereomers of compound 1, compound 2
was obtained by filtration quantitatively. Then, primary alcohol
was protected by the treatment of 4,40-dimethoxytrityl chloride
and catalytic amount of dimethylaminopyridine in pyridine at
50 °C for 2 h. The total yield of two steps was 47%. The phospho-
ramidite 4 was obtained from compound 3, reacting with chloro-
(2-cyanoethoxy)-N,N-diisopropylaminophosphine in 4-methyl-
morpholine and CH2Cl2 for 5 min. All compounds were character-
ized by 1H, 13C, 31P NMR, and mass spectrometry.
The phosphoramidite 4 was firstly introduced in the middle of
the oligodeoxyribonucleotide (ODN) to determine its property as
a hairpin loop. We synthesized four hairpin ODNs by oligonucleo-
tide synthesizer. The modifier 4 could easily couple with natural
sequences under the normal conditions and the synthesized oligo-
nucleotides were characterized by MALDI-MS.15
Table 1 shows the Tm values of hairpin ODNs compared with
those of natural one. Compound 4 and natural loop unit, C4, were
inserted in the middle of the palindromic DNA sequences. The Tm
value of A4T was higher than that of AC4T. It means that A4T
had more stable structure than AC4T. In case of Y1XY1, when the
stem length was just six base parings, Tm of Y14Y1 was slightly low-
er than the natural one, Y1C4Y1. However, Y24Y2 containing seven
base pair stem formed stable hairpin by introducing the compound
Hairpin structure is the basic secondary structure in nucleic
acid world. Because of their high stability, intramolecular hairpin
structure with various loop units has been reported. Introducing
foreign loop molecules, intramolecular structures showed better
properties such as structural stability7, nuclease resistance8, elec-
tron transfer9, structural reversibility10, and therapeutic ability11
.
We previously reported the hairpin oligonucleotide bearing
cholane-3,24-diol moiety as a loop.12 In a similar manner, we in-
serted non-nucleosidic molecule at the loop position and synthe-
sized hairpin oligonucleotide. The loop molecule was derived
from 18b-gycyrrhetinic acid, which is one of the components of ex-
* Corresponding author. Tel.: +82 54 279 2115; fax: +82 54 279 3399.
E-mail address: bhkim@postech.ac.kr (B.H. Kim).
4 in the loop position, accompanied with much higher
DTm value
0040-4039/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2009.03.049

2546
E.-K. Bang, B. H. Kim / Tetrahedron Letters 50 (2009) 2545–2547
Scheme 1. Reagents and conditions: (a) LiAlH4, THF, 60 °C, 4 h, 95%; (b) Pt/C(10 wt %), H2 (1 atm), CHCl3/THF(1/1), rt, 2 h; (c) 4,40-dimethoxytrityl chloride,
dimethylaminopyridine, pyridine, 45–50 °C, 2 h, 47% (2 steps); (d) 4-methylmorpholine, chloro-(2-cyanoethoxy)-N,N-diisopropylaminophosphine, CH2Cl2, rt, 5 min, 81%.
Table 1
Table 2
Sequence of ODNs and their Tm values
Sequence of synthesized shRNAs
Name
Sequence (50–30)
Tm (°C)
Name
Sequence
X = 4
X = C4
AXT
d(A12 X T12
d(AACGTT X AACGTT)
d(CAA CGT T X A ACG TTG)
d(CC AAC GTT X AA CGT TGG)
)
73
63
75
80
68
66
—
Y1XY1
Y2XY2
Y3XY3
L-C4
L-4
—
Tm values were determined by measuring changes in absorbance at 260 (cuvette,
1 cm path length) as a function of temperature in Tris–HCl buffer (10 mM, pH 7.2)
containing NaCl (100 mM) and MgCl2 (20 mM). The temperature was raised at a
rate of 1.0 °C/min. Total ODN concentration is 3 lM.
between Y14Y1, and Y24Y2 than between Y24Y2, and Y34Y3. In com-
parison with our previous data of another stable loop modifier (Tm
values were 72, 76, and 82 °C for Y1XY1, Y2XY2, and Y3XY3 respec-
tively.),12 Y24Y2 has minimum stem length to form highly stable
hairpin structure.
Modification of the loop position could not disturb their whole
structures as illustrated in Figure 1. These are CD spectra of A4T
and AC4T. These two ODNs had nearly similar patterns of curves,
representing positive bands at 218 and 283–284 nm and a negative
band at 248–249 nm. These CD spectra were almost identical to
that of known B-form duplex structures.
S-C4
S-4
By using this hairpin loop mimic compound 4, we synthesized
shRNAs as shown in Table 2. We incorporated compound 4 and
natural hairpin loop unit, C4, between sense and antisense strand.
Our synthesized shRNAs are classified into two groups, L-group
and S-group. L-group has longer linker than S-group between loop
and siRNA sequences. The shRNAs were synthesized by oligonu-
cleotide synthesizer and purified by HPLC.
As a target of all shRNAs, EGFP gene was used to determine their
interfering efficiency by measuring fluorescence of remained GFP.
The EGFP suppression assay using synthesized shRNAs was done
in collaboration with Dr. Dong-Ho Kim (Beckman Research Insti-
tute of the City of Hope). The experimental protocols were exactly
similar to their previous methods except for using NIH3T3 cell line
here.16 If the anti-EGFP shRNAs worked through RNAi mechanism,
reduced fluorescence would be detected because GFP was
downregulated.
After transfection with Lipofectamine 2000 (invitrogen), all syn-
thetic RNAs showed highly reduced fluorescence signal comparing
with control treatment without shRNA (Fig. 2). It suggests that
modified shRNAs have potent activity as a gene silencer and chang-
ing loop position with foreign molecule does not suppress their
Figure 1. CD spectra of A4T and AC4T. The curves were obtained at 10 °C and the
condition was as shown in Table 1.

E.-K. Bang, B. H. Kim / Tetrahedron Letters 50 (2009) 2545–2547
2547
2. (a) Venkatesan, N.; Kim, S. J.; Kim, B. H. Curr. Med. Chem. 2003, 10, 1973–1991;
(b) Kim, S. J.; Kim, B. H. Nucleic Acids Res. 2003, 31, 2725–2734; (c) Kim, S. J.;
Kim, B. H. Nucleosides Nucleotides Nucleic Acids 2003, 22, 2003–2011; (d) Kim, S.
J.; Bang, E.-K.; Kim, B. H. Synlett 2003, 1838–1840; (e) Hwang, G. T.; Seo, Y. J.;
Kim, S. J.; Kim, B. H. Tetrahedron Lett. 2004, 45, 3543–3546; (f) Hwang, G. T.;
Seo, Y. J.; Kim, B. H. J. Am. Chem. Soc. 2004, 126, 6528–6529; (g) Hwang, G. T.;
Seo, Y. J.; Kim, B. H. Tetrahedron Lett. 2005, 46, 1475–1477; (h) Seo, Y. J.; Ryu, J.
H.; Kim, B. H. Org. Lett. 2005, 7, 4931–4933; (i) Seo, Y. J.; Kim, B. H. Chem.
Commun. 2006, 150–152; (j) Seo, Y. J.; Hwang, G. T.; Kim, B. H. Tetrahedron Lett.
2006, 47, 4037–4039; (k) Seo, Y. J.; Jeong, H. S.; Bang, E.-K. Bioconjugate Chem.
2006, 17, 1151–1155; (l) Ryu, J. H.; Seo, Y. J.; Hwang, G. T.; Lee, J. Y.; Kim, B. H.
Tetrahedron 2007, 63, 3538–3547; (m) Seo, Y. J.; Rhee, H.; Joo, T.; Kim, B. H. J.
Am. Chem. Soc. 2007, 129, 5244–5247; (n) Seo, Y. J.; Lee, I. J.; Yi, J. W.; Kim, B. H.
Chem. Commun. 2007, 2817–2819; (o) Venkatesan, N.; Seo, Y. J.; Kim, B. H.
Chem. Soc. Rev. 2008, 37, 648–663; (p) Seo, Y. J.; Lee, I. J.; Kim, B. H. Bioorg. Med.
Chem. Lett. 2008, 18, 3910–3913.
3. Fire, A.; Xu, S.; Montgomery, M. K.; Kostas, S. A.; Driver, S. E.; Mello, C. Nature
1998, 391, 806–811.
Figure 2. RNA interference efficacy test: all shRNAs were annealed in sodium
phosphate buffer (20 mM, pH 6.8) containing NaCl (100 mM).
4. (a) Henry, C. M. Chem. Eng. News 2003, 81, 32–36; (b) Rana, T. M. Nat. Mol. Cell
Biol. 2007, 8, 23–36; (c) Paddison, P. J.; Caudy, A. A.; Bernstein, E.; Hannon, G. J.;
Conklin, E. S. Genes Dev. 2002, 16, 948–958; (d) Kurreck, J. Angew. Chem., Int. Ed.
2009, 48, 1378–1398; (e) Jeong, J. H.; Mok, H.; Oh, Y.-K.; Park, T. G. Bioconjugate
Chem. 2009, 20, 5–14.
RNAi activity. S-4 had higher RNAi efficacy than L-4, but this lin-
ker-dependent phenomenon was not applicable to natural one.
In conclusion, we synthesized a new building block which was
introduced into the oligonucleotide to function as a hairpin loop.
The hairpin oligonucleotides with this rigid molecule formed sta-
ble hairpin systems and more than seven base-paired stems were
needed to have higher Tm value than natural one. We could observe
that the novel loop modifier did not distort original duplex struc-
ture. The shRNAs bearing derivative of 18b-glycyrrhetinic acid
could interfere with their target gene expression without affecting
their RNAi efficiency, by carefully designing the linker sequences
between the loop and siRNA part. It indicates that we can modify
the loop position of shRNA with not only nucleotide but also with
any possible molecule such as fluorophores, membrane permeable
molecules, and receptor binding molecules.
5. Brummelkamp, T. R.; Bernards, R.; Agami, R. Science 2002, 296, 550–553.
6. (a) Hoshika, B. S.; Minakawa, N.; Matsuda, A. Nucleic Acids Res. 2004, 32, 3815–
3825; (b) Chiu, Y.-L.; Rana, T. M. RNA 2003, 9, 1034–1048; (c) Qin, X.-F.; An, D.
S.; Chen, I. S. Y.; Baltimore, D. Nucleic Acids Res. 2003, 31, 589–595; (d) Hamada,
M.; Ohtsuka, T.; Kawaida, R.; Koizumi, M.; Morita, K.; Furukawa, H.; Imanishi,
T.; Miyagishi, M.; Taira, K. Antisense Nucleic Acid Drug Dev. 2002, 12, 301–309;
(e) Paula, D. D.; Vitoria, M.; Bentley, L. B.; Mahato, R. I. RNA 2007, 13, 341–348;
(f) Santel, A.; Aleku, M.; Keil, O.; Endruschat, J.; Esche, V.; Fisch, G.; Dames, S.;
Löffler, K.; Fechtner, M.; Arnold, W.; Giese, K.; Klippel, A.; Kaufmann, J. Gene
Ther. 2006, 13, 1222–1234; (g) Lorenz, C.; Hadwiger, P.; John, M.; Vornlocher, H.
P.; Unverzagt, C. Bioorg. Med. Chem. Lett. 2004, 14, 4975–4977; (h) Soutschek, J.;
Akinc, A.; Bramlage, B.; Charisse, K.; Constien, R.; Donoghue, M.; Elbashir, S.;
Geick, A.; Hadwiger, P.; Harborth, J.; John, M.; Kesavan, V.; Lavine, G.; Pandey,
R. K.; Racie, T.; Rajeev, K. G.; Röhl, I.; Toudjarska, I.; Wang, G.; Wuschko, S.;
Bumcrot, D.; Koteliansky, V.; Limmer, S.; Manoharan, M.; Vornlocher, H.-P.
Nature 2004, 432, 173–178; (i) Wolfrum, C.; Shi, S.; Jayaprakash, K. N.;
Jayaraman, M.; Wang, G.; Pandey, R. K.; Rajeev, K. G.; Nakayama, T.; Charrise,
K.; Ndungo, E. M.; Zimmermann, T.; Koteliansky, V.; Manoharan, M.; Stoffel, M.
Nat. Biotechnol. 2007, 25, 1149–1157.
7. Stutz, A.; Langenegger, S. M.; Häner, R. Helv. Chim. Acta 2003, 86, 3152–3163.
8. (a) Tarköy, A.; Phipps, A. K.; Schultze, P.; Feigon, J. Biochemistry 1998, 37, 5810–
5819; (b) Zhang, L.; Long, H.; Schatz, G. C.; Lewis, F. D. Org. Biomol. Chem. 2006,
5, 450–456.
9. (a) Lewis, F. D.; Letsinger, R. L.; Wasielewski, M. R. Acc. Chem. Res. 2001, 34,
159–170; (b) Beljonne, D.; Pourtois, G.; Ratner, M. A.; Brédas, J. L. J. Am. Chem.
Soc. 2003, 125, 14510–14517.
10. Lewis, F. D.; Wu, Y.; Zhang, L. Chem. Commun. 2004, 636–637.
11. Nakane, M.; Ichikawa, S.; Matsuda, A. J. Org. Chem. 2008, 73, 1842–1851.
12. (a) Kim, S. J.; Bang, E. K.; Kwon, H. J.; Shim, J. S.; Kim, B. H. ChemBioChem 2004,
5, 1517–1522; (b) Venkatesan, N.; Seo, Y. J.; Bang, E.-K.; Park, S. M.; Lee, Y. S.;
Kim, B. H. Bull. Kor. Chem. Soc. 2006, 27, 613–630.
Acknowledgments
We thank Dr. Dong-Ho Kim for his contribution on the EGFP
suppression assay in Beckman Research Institute of the City of
Hope in the USA. This work has been supported by KOSEF (Gene
Therapy R&D and KNRRC programs).
Supplementary data
13. Um, S.-J.; Park, M.-S.; Park, S.-H.; Han, H.-S.; Kwon, Y.-J.; Sin, H.-S. Bioorg. Med.
Chem. 2003, 11, 5345–5352.
14. Shibata, S.; Takahashi, K.; Yano, S.; Harada, M.; Saito, H.; Tamura, T.; Kumagai,
A.; Hirabayashi, K.; Yamamoto, M.; Nagata, N. Chem. Pharm. Bull. 1987, 35,
1910–1918.
15. MALDI-TOF MS data of hairpin ODNs: A4T 7852.7 (calcd 7855.5); Y14Y1 4150.2
(calcd 4150.3); Y24Y2 4768.3 (calcd 4768.6); Y34Y3 5388.7 (calcd 5387.0).
16. Kim, D.-H.; Behlke, M. A.; Rose, S. D.; Chang, M.-S.; Choi, S.; Rossi, J. J. Nat.
Biotechnol. 2005, 23, 222–226.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.tetlet.2009.03.049.
References and notes
1. (a) Uhlmann, E.; Peyman, A. Chem. Rev. 1990, 90, 543–584; (b) Kool, E. T. Acc.
Chem. Res. 1998, 31, 502–510.