Modified GSMP synthesis greatly improves the disulfide crosslink of T7 run-off siRNAs with cell penetrating peptides

Article abstract of DOI:10.1055/s-0030-1259022

Preventing intramolecular cyclization greatly improves 5′-deoxy- 5′-thioguanosine monophosphorothioate (GSMP) synthesis and its application as a potent initiator nucleotide for T7 run-off transcription of noncoding RNAs. GSMP was efficiently incorporated into the 5′-end of siRNA sense strands and the resulting thiol-modified siRNA was crosslinked with a free cysteine of cell penetrating peptide Penetratin as monitored by mass spectrometry. Cellular uptake and the knockdown potential of the peptide-coupled siRNA (pepsiRNA) were evaluated in primary cells. Georg Thieme Verlag Stuttgart - New York.

Full text of DOI:10.1055/s-0030-1259022

LETTER
2959
Modified GSMP Synthesis Greatly Improves the Disulfide Crosslink of T7
Run-Off siRNAs with Cell Penetrating Peptides
GSMP Synthesis
f
a
or T7 Run-
t
Off siR
j
N
As a Schmitz,a,b Frank Hahn,a,c Ute Schepers*a,d,e
a
Kekulé-Institut für Organische Chemie und Biochemie, Rheinische Friedrich-Wilhelms Universität Bonn,
Gerhard-Domagk-Str.1, 53121 Bonn, Germany
b
c
d
e
Institute of Organic Chemistry, Karlsruhe Institute of Technology (KIT), Fritz-Haber-Weg 6, 76131 Karlsruhe, Germany
Department of Biochemistry, University of Cambridge, Downing Site, Cambridge CB2 1QW, U.K.
Institute of Applied Biosciences, Karlsruhe Institute of Technology (KIT), Herzstr. 16, 76187 Karlsruhe, Germany
Institute of Toxicology and Genetics, Karlsruhe Institute of Technology (KIT), Hermann-von-Helmholtz Platz 1,
PO Box 3640, 76021 Karlsruhe, Germany
Fax +49(7247)823354; E-mail: ute.schepers@kit.edu
Received 31 July 2010
tively incorporated at the 5¢-terminus.7 GMP derivatives
are favored as guanosine is the best enhancer of the initi-
ation rate of T7 in vitro transcription. RNA modified with
guanosine monophosphorothioate (GMPS) can be direct-
Abstract: Preventing intramolecular cyclization greatly improves
5¢-deoxy-5¢-thioguanosine monophosphorothioate (GSMP) synthe-
sis and its application as a potent initiator nucleotide for T7 run-off
transcription of noncoding RNAs. GSMP was efficiently incorpo-
rated into the 5¢-end of siRNA sense strands and the resulting thiol- ly coupled with thiopyridyl-activated compounds8 to form
less stable phosphorothioate sulfides (R-SSPO3-RNA).9
modified siRNA was crosslinked with a free cysteine of cell pene-
trating peptide Penetratin as monitored by mass spectrometry. Cel-
Conversely, modification with 5¢-deoxy-5¢-thioguanosine
lular uptake and the knockdown potential of the peptide-coupled
monophosphorothioate (GSMP)4a leads to a 5¢-terminal
siRNA (pepsiRNA) were evaluated in primary cells.
phosphate, which needs to be removed by a phosphatase
Key words: guanosine, T7 RNA, siRNA, Penetratin, disulfide
crosslink, knockdown
to liberate the thiol function. The resulting 5¢-HS-RNA
forms more stable disulfide bonds upon coupling. At the
same time this approach provides a minimal distance be-
tween the cargo molecule and the RNA (R-S-S-RNA).
Over the past decade, RNA-based techniques such as an-
The preparation of GSMP has been described as a four-
tisense-mediated silencing,1 RNA-aptamers,2 and RNA
step synthesis (Scheme 1).4a However, the fast intramo-
interference (RNAi)3 have become important tools for
lecular cyclization of the iodinated isopropylidenegua-
regulation of gene function. Thus, there is a great interest
in noncoding RNA functionalization in order to immobi-
lize RNA to solid phase for in vitro evolution or to co-
nosine 3 leads to a severe decrease in yields of the
unprotected 5¢-desoxyguanosine. Another route to 5¢-
deoxy-5¢-iodoguanosine, which is stable against this side
valently couple reporter molecules, ligands for targeting
reaction, is based on the iodination of the 5¢-position with-
out the protection of the 3¢- and 2¢-OH groups.10 Although
this synthesis has been described in several examples with
yields in the range of 70%, it suffers from the difficulty to
isolate a pure product. In our approach we introduced an
additional protection step to reduce the rate of the side re-
action and used the GSMP in vitro transcription strategy
to generate 5¢-SH-functionalized siRNAs. Finally, the
overall yield of 5¢-desoxy-5¢-iodoguanosine is improved
compared to the other approaches. Furthermore, the for-
mation of susceptible intermediates or purification prob-
lems is avoided. Following the previously reported
synthesis4a guanosine (1) was protected at its 2¢- and 3¢-
hydroxy groups by an acetal to produce 2¢,3¢-O,O-isopro-
pylideneguanosine (2) in 94% yield. Reaction of the 5¢-
hydroxy group of 2 with methyltriphenoxyphosphonium
iodide (MTPI) in a Moffat reaction11 afforded the crude
5¢-deoxy-5¢-iodo-2¢,3¢-O,O-isopropylideneguanosine (3)
as a sticky paste leading to a yield of only 39%. However,
2¢,3¢-O,O-isopropylidene-N-3,5¢-C-cycloguanosine (6)
was formed as a by-product to 3. This type of cyclization
has been described previously for related compounds
(Scheme 1).11,12 To prevent the cyclization and to stabilize
the product, we protected the primary amino group by for-
or delivery vectors as functional molecules.4 Especially,
the transfection of certain mammalian cell lines and pri-
mary cells with RNAs can be cumbersome. Previously,
cell-penetrating peptides (CPPs) were coupled to siRNAs
to enhance cellular uptake.5
For this kind of delivery strategy the functionalization of
the RNA with thiol groups is particularly attractive since
they react with the thiol group of cysteine-modified CPPs
under the formation of disulfide bonds.6 Once these si-
RNA-conjugates reach the interior of a cell, the disulfide
bonds are cleaved under the reducing conditions of the cy-
tosol and siRNA is released from the delivery peptide. In
our approach, we chose an enzymatic route to thiol-mod-
ified siRNAs for the coupling to CPPs. This strategy can
be adapted to the peptide coupling of other noncoding
RNAs. If RNA is transcribed from a DNA template with
T7 RNA polymerase, the 5¢-terminus of the RNA can be
modified by the use of guanosine monophosphate (GMP)
derivatives that act as initiator nucleotides and are selec-
SYNLETT 2010, No. 19, pp 2959–2963
x
x
.x
x
.2
0
1
0
Advanced online publication: 03.11.2010
DOI: 10.1055/s-0030-1259022; Art ID: G25010ST
© Georg Thieme Verlag Stuttgart · New York

2960
K. Schmitz et al.
LETTER
mation of an imine with dimethylformamide dimethyl ac- O,O-isopropylidene-5¢-deoxy-5¢-iodoguanosine (9) in
etal,13 which reduced the flexibility of the purine and 99% yield.14 The dried compound 9 could be stored for
thereby the rate of intramolecular nucleophilic attack. four weeks at 4 °C without intramolecular cyclization.
2-N,N-Dimethylaminomethylene-2¢,3¢-O,O-isopropylidene-
The rate of cyclization for the unprotected 3 and protected
guanosine (8) was obtained in 80% yield and could be io-
iodide 9 was monitored by 1H NMR in DMSO-d6 at am-
dinated to yield 2-N,N-dimethylaminomethylene-2¢,3¢-
bient temperature (Figure S1, Supporting Information).
1
For 3, H NMR spectra were recorded every 30 minutes
revealing a first order kinetic with k = 0.181 0.008 h–1
O
1
N
N
while recording H NMR spectra for protected iodide 9
NH
every 24 hours yielded a first-order rate constant of k =
0.0145 0.0008 h–1 corresponding to an approximately
12-fold decrease in reaction rate (Figure 1).
N
NH2
HO
O
HO
OH
N
O
N
1
O
N
N
NH
NH
a)
N
N
N
NH2
Me
HO
HO
N
O
O
e)
Me
O
O
O
O
8
O
N
O
N
2
N
N
N
N
NH
NH
f)
b)
NH2
N
Me
I
I
N
O
Figure 1 Introduction of an N,N-dimethylaminomethylene protec-
ting group increased the half life time of 3 by a factor of 12. Averages
of significant integrals for 9 (dashed) and unprotected 3 (solid) are
plotted against time. Both sets of data are fitted with a first-order
kinetic with k = 0.0145 h–1 and k = 0.181 h–1, respectively.
O
Me
O
O
O
O
9
3
Both protecting groups, the acetal and the imine, could be
removed from 9 by stirring in 50% formic acid for three
days at room temperature yielding 5¢-deoxy-5¢-iodogua-
nosine (4) without formation of the cyclization product 7.
Compound 4 was treated with a 2–3-fold molar excess of
sodium thiophosphate in degassed water under argon to
yield 5¢-deoxy-5¢-thioguanosine monophosphorothioate
g)
c)
O
O
O
N
I
H
N
N
N
HN
N
N
NH
3
N
H2N
N
H2N
N
N
9
NH2
I
I
O
O
5'
O
1'
O
O
O
O
HO
OH
4
3
6
d)
– HI
O
N
O
N
N
N
NH
N
N
O
NH2
N
–O
P
S
H2N
O–
O
O
OH
HO
O
O
5
7
Scheme 1 Modification of GSMP synthesis.7 Reagents and conditi-
ons: (a) acetone, cat. HClO4, 4 h, r.t.; (b) MTPI, THF, 30 min, –65 °C,
4 h, r.t.; (c) 50% HCOOH, 3 d; (d) Na3PO3S, H2O, 3 d, r.t.; cyclization
of 3 generates 2¢,3¢-O,O-isopropylidene-3,5¢-C-cycloguanosine (7)
via a charged intermediate (6).11 Modified synthesis of GSMP.
Reagents and conditions: (e) (MeO)2HCNMe2, DMF, 4 h, 50 °C; (f)
MTPI, THF, 30 min, –65 °C, 4 h, r.t.; (g) 50% HCOOH.
Figure 2 Schematic view of the enzymatic synthesis of thiol-modi-
fied siRNAs and their coupling to the cell penetrating peptide Pene-
tratin
Synlett 2010, No. 19, 2959–2963 © Thieme Stuttgart · New York

LETTER
GSMP Synthesis for T7 Run-Off siRNAs
2961
(GSMP; 5). Yields could be greatly improved by dissolv- SH-siRNAs were incubated with dithiodipyridyl-activat-
ing the thiophosphate in degassed water and removing the ed Penetratin to form conjugates in a thiol-exchange reac-
1
precipitate prior to the addition to a suspension of 4. H tion to selectively form heterodimers.15
NMR and 31P NMR spectroscopy indicated quantitative
formation of GSMP (5) (d = 19 ppm) (Figure S1, Support-
The ratio of modified to unmodified RNA was determined
by a gel shift assay (see Supporting Information). The re-
ing Information). After precipitation of the remaining
sulting peptide-coupled siRNAs (pepsiRNAs) were char-
thiophosphate the crude product was purified by gravity
acterized by MALDI mass spectrometry and SDS-PAGE
chromatography on RP-18 with water to afford pure
under reducing and non-reducing conditions (Figure 3).
GSMP in 68% yield. To prove its suitability as an initiator
Finally, the enzymatically prepared pepsiRNAs were test-
nucleotide, purified GSMP was then used for the enzy-
ed for their ability to mediate RNAi in mammalian cell
culture in comparison to conjugates of synthetic thiolated
siRNA with Penetratin. Murine primary fibroblasts and
myogenic cells were obtained from a transgenic GFP
matic synthesis of 5¢-thiolated siRNAs (Figure 2).
SiRNAs were generated by T7-based in vitro transcription
of DNA oligonucleotides encoding the appropriate 21-nt
sequences of the respective target genes. The DNA tem-
mouse,16 and down-regulation of GFP upon treatment
plates comprised a T7 recognition sequence at the 5¢-end,
with 10–25 nM pepsiRNAs was measured by live micros-
copy after 48 hours (Figure 4). Both synthetic 5¢-SH-C6-
modified siRNA and the enzymatically generated siRNA
which was hybridized with a matching oligonucleotide to
provide a partially double-stranded recognition site for
T7-polymerase. In vitro transcription led to RNA-
induced a knockdown of GFP to about 35%.
transcripts complementary to the single stranded DNA-
sequence containing a guanosine at the 5¢-end and a 2-nt
3¢-TT overhang. For the generation of 5¢-thiol-modified
siRNAs an eight-fold excess of GSMP (5) was added to
the sense strand reaction mixture. Following transcrip-
tion, hybridization, and treatment with DNase to degrade
residual DNA template and calf intestinal phosphatase to
remove the 5¢-phosphate of the GSMP, the disulfide-
linked dimers were reduced with DTT. Eventually, the 5¢-
Figure 4 RNA interference by synthetic and enzymatic pepsiRNAs.
Primary myogenic cells from skeletal muscle (a–c) and primary mu-
rine fibroblasts (d–f) of transgenic GFP mice were treated with 10 nM
(b) and 25 nM (c) of pepsiRNA (synthetic) against GFP and 10 nM
(e, f) of pepsiRNA (enzymatic). Fluorescent micrographs were taken
72 h post treatment using the appropriate GFP/FITC filter. (f) The da-
shed line shows the surrounding of the cells that exhibit a knockdown
Figure 3 MALDI and SDS-PAGE analysis of siRNA-Penetratin
conjugate. (a) MALDI spectrum of pepsiRNA (GFP) duplex before
and (b) after reduction with DTT. The spectra (negative mode, matrix:
THAP, NH4Ac added) show four peaks corresponding to ss-siRNA
(ca. 7kD), pepsiRNA (sense strand) (ca. 9.8 kD), siRNA duplex (ca.
14 kD) and pepsiRNA duplex (ca. 16.8 kD, ca. 8.4 kD). (c) 15%-SDS-
PAGE analysis of Penetratin (left panel), pepsiRNAs derived from a
synthetic 5¢-SH-C6-siRNA (right panel) and the corresponding enzy-
matically synthesized 5¢-SH-siRNA (middle panel) under non-redu-
cing and reducing conditions.
of GFP.
We also synthesized pepsiRNAs against glucosylcer-
amide synthase (GCS), an endogenous gene, which is in-
volved in the synthesis of glucosylceramide (GlcCer).
GlcCer is essential for the transport of tyrosinase to the
melanosomes, where it converts tyrosine into L-DOPA in
the first rate-limiting step of melanine synthesis and pig-
Synlett 2010, No. 19, 2959–2963 © Thieme Stuttgart · New York

2962
K. Schmitz et al.
LETTER
(2) Famulok, M. Curr. Opin. Struct. Biol. 1999, 9, 324.
(3) (a) Arenz, C.; Schepers, U. Naturwissenschaften 2003, 90,
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2004, 24, 10040. (b) Muratovska, A.; Eccles, M. R. FEBS
Lett. 2004, 558, 63.
mentation.17 Down-regulation of pigmentation was moni-
tored as an indicator of GCS silencing 2–4 days after
treatment of murine melanoma cells. Knockdown of GCS
to below 10% was determined by mRNA quantification
and by Western blot analysis, and could be observed as a
loss of pigmentation in melanoma cells (Figure 5). These
model experiments show that our alternative strategy
leads to functional peptide-coupled siRNAs that are suit-
able for the treatment of primary mammalian cells.
(6) Fischer, P. M.; Krausz, E.; Lane, D. P. Bioconjugate Chem.
2001, 12, 825.
(7) (a) Seelig, B.; Jäschke, A. Tetrahedron Lett. 1997, 38, 7729.
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S.; Jäschke, A.; Famulok, M. Bioorg. Med. Chem. 2000, 8,
1317. (b) Goody, R. S.; Walker, R. T. J. Org. Chem. 1971,
36, 727.
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(11) Verheyden, J. P.; Wagner, D.; Moffatt, J. G. J. Org. Chem.
1970, 35, 2319.
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W. J.; Wagner, G.; Halperin, J. A. Bioorg. Med. Chem. Lett.
2004, 14, 2657.
Figure 5 RNAi with pepsiRNA(GCS) in murine melanoma cells.
(a) pepsiRNA (5–25 nM) against GCS was added to MEB4 cells. As
controls, cells were treated with 100 nM uncoupled Penetratin. The
data were averaged from five independent experiments +/– s.d. GCS
mRNA levels were measured by RT-PCR (a) as well as by Western
blot (for 25 nM pepsiRNA) (b). Ratios were normalized to the non-
treated cells. (c) Visual assay: MEB4 cells (1 × 107) were treated with
the pepsiRNA (25 nM) against GCS. The color of the harvested cells
shows a lack of pigmentation in GCS-deficient cells.
(14) N,N-Dimethylaminomethylene-2¢,3¢-O,O-isopro-
pylideneguanosine (8): 2¢,3¢-O,O-Isopropylideneguanosine
(2; 5.75 g, 17.8 mmol) was resuspended in DMF (60 mL)
and N,N-dimethylformamide dimethyl acetal (8.91 mL) was
added under argon to yield an orange-brown solution. The
reaction mixture was stirred at 50 °C for 4 h. The solvent was
removed under reduced pressure and at elevated
temperatures (ca. 55 °C), the white residue was then
removed by filtration. The filtrate was dried under reduced
pressure, redissolved in MeOH (25 mL) yielding a
fluorescent green solution and precipitated with 50 mL of
EtOAc. After storage overnight at 4 °C, the residue was
removed by filtration and the combined filtrates were
thoroughly washed with EtOAc and dried under reduced
pressure. The product was obtained as a white powder in
80% yield (5.38 g, 14.2 mmol). All steps were carried out
under protection from light; mp >200 °C; Rf 0.47 (CHCl3–
MeOH, 8:1), 0.08 (CHCl3–MeOH, 19:1). 1H NMR (400
MHz, DMSO-d6): d = 11.32 (br s, 1 H, NH), 8.56 (s, 1 H, H-
imine), 8.00 (s, 1 H, H-8), 6.03 (d, J = 3.0 Hz, 1 H, H-1¢),
5.26 (dd, J = 3.0, 6.3 Hz, 1 H, H-2¢), 5.03 (dd, J = 5.4, 5.4
Hz, 1 H, 5¢-OH), 4.95 (dd, J = 2.9, 6.3 Hz, 1 H, H-3¢), 4.13
(ddd, J = 2.9, 4.9, 4.9 Hz, 1 H, H-4¢), 3.59–3.47 (m, 2 H, H-
5¢), 3.15 (s, 3 H, NMe), 3.03 (s, 3 H, NMe), 1.53 (s, 3 H, Me),
1.33 (s, 3 H, Me). 13C NMR (101 MHz, DMSO-d6): d =
158.3 (C-imine), 157.6, 157.5 (C-4, C-6), 149.6 (C2), 136.0
(C-8), 119.9 (C-5), 113.2 (C-quat), 88.6, 86.4, 83.6, 81.2 (C-
1¢ to C-4¢), 61.5 (C-5¢), 40.8 (NMe), 34.8 (NMe), 27.2 (Me),
25.3 (Me). MS (EI; 70 eV): m/z (%) = 378 (89%) [M]+, 363
(6%) [M – CH]+, 348 (6%) [M – 2 Me]+, 333 (2%) [M –
NH(Me)2]+, 206 (100%) [M – DAMG]+, 191 (26%) [DAMG
– Me]+, 176 (3%) [DAMG – 2 Me]+, 150 (6%) [guanine]+.
HRMS: m/z calcd for C16H22N6O5: 378.1652; found:
378.1654 ( 0.0095); (DAMG = 2-N¢,N-
In conclusion, we have demonstrated an alternative route
to peptide-coupled noncoding RNAs using GSMP as an
initiator nucleotide in T7 in vitro transcription. As an ex-
ample we generated siRNAs with a thiol modification for
disulfide crosslinking with a cell penetrating peptide. The
so-called pepsiRNAs have been shown to be suitable for
their use in RNAi experiments. The strategy was devel-
oped due to shortcomings in the synthesis of GSMP. For-
mation of cyclization by-products and lack of solubility of
the precursor reduce the overall yield. The synthesis was
greatly improved by the introduction of an imine protect-
ing group on the purine base preventing intramolecular
cyclization. With an optimized purification protocol for
GSMP we achieved an overall yield of 53% in five steps
as opposed to the reported 35% in four steps.4a
Supporting Information for this article is available online at
http://www.thieme-connect.com/ejournals/toc/synlett.
Acknowledgment
We would like to thank Prof. Dr. S. Bräse for scientific discussions,
G. Weyand for help with NMR measurements, and Dr. Rustam
Mundegar for the primary transgenic GFP cells from GFP mice.
The work was financially supported by the DFG Center for Functio-
nal Nanostructures (CFN), Karlsruhe, the Fonds der Chemischen
Industrie and the SFB645, Project Z1.
dimethylaminomethylene guanine).
N,N-Dimethylaminomethylene-2¢,3¢-O,O-isopro-
pylidene-5¢-deoxy-5¢-iodoguanosine (9): Ground 8 (2.20 g,
References and Notes
(1) Brantl, S. Biochim. Biophys. Acta 2002, 1575, 15.
Synlett 2010, No. 19, 2959–2963 © Thieme Stuttgart · New York

LETTER
5.81 mmol) was resuspended in anhyd THF (110 mL) under
GSMP Synthesis for T7 Run-Off siRNAs
2963
J = 5.7, 9.9 Hz, 1 H, H-5¢), 3.19 (s, 3 H, NMe), 3.04 (s, 3 H,
NMe), 1.53 (s, 3 H, Me), 1.35 (s, 3 H, Me). 13C NMR (101
MHz, DMSO-d6): d = 158.2 (C-imine), 157.7, 157.5 (C-4,
C-6), 149.2 (C2), 137.9 (C-8), 120.2 (C-5), 113.5 (C-quart),
89.4, 86.4, 84.0, 83.9 (C-1¢ to C-4¢), 41.1 (NMe), 34.9
(NMe), 27.2 (Me), 25.3 (Me), 6.7 (C-5¢). MS (EI; 70 eV):
m/z = 488 (13%) [M]+, 361 (7%) [M – I]+, 360 (35%)
[M – HI]+, 345 (1%) [M – HI, Me]+, 206 (3%) [DAMG]+,
188 (2%) [DAMG – H2O]+, 176 (26%) [DAMG – 2 Me]+,
149 (12%) [guanine – H]+, 128 (100%) [HI]. HRMS: m/z
calcd for C16H21N6O4I: 488.0669; found: 488.06631;
(DAMG = 2-N¢,N-dimethylaminomethylene guanine).
(15) Zhang, L.; Sun, L. L.; Cui, Z. Y.; Gottlieb, R. L.; Zhang,
B. L. Bioconjugate Chem. 2001, 12, 939.
argon and cooled to –70 °C by a mixture of acetone and dry
ice. Methyltriphenoxyphosphonium iodide (3.94 g, 8.71
mmol; 1.5 equiv) was added. Due to the light sensitivity of
the reactant and the product all subsequent steps were carried
out under exclusion of light. After 30 min of stirring the
reaction mixture was allowed to warm to r.t. and stirred for
another 4 h. The reaction was stopped by the addition of
MeOH (5 mL) and the solvent was removed under reduced
pressure. The dark red residue was dissolved in MeOH–
CHCl3 (1:4; 2.5 mL) and CHCl3 (7 mL) was added. The
solution of the crude product was subjected to chromatog-
raphy on silica gel (Merck 0.40–0.65 mm; eluent: CHCl3–
MeOH, 9:1). After removal of the solvent, 9 was obtained as
an orange solid in 99% yield (2.81 g, 5.75 mmol); mp 95–97
°C; Rf 0.74 (CHCl3–MeOH, 5:1), 0.22 (CHCl3–MeOH,
19:1). 1H NMR (400 MHz, DMSO-d6): d = 11.39 (br s, 1 H,
NH), 8.58 (s, 1 H, H-imine), 8.01 (s, 1 H, H-8), 6.15 (d, J =
2.1 Hz, 1 H, H-1¢), 5.42 (dd, J = 2.1, 6.3 Hz, 1 H, H-2¢), 5.02
(dd, J = 2.8, 6.3 Hz, 1 H, H-3¢), 4.28 (ddd, J = 2.8, 5.7, 9.0
Hz, 1 H, H-4¢), 3.49 (dd, J = 9.0, 9.9 Hz, 1 H, H-5¢), 3.30 (dd,
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Klumperman, J.; van der Sluijs, P.; van Meer, G. J. Cell
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Synlett 2010, No. 19, 2959–2963 © Thieme Stuttgart · New York