A versatile post-synthetic method on a solid support for the synthesis of RNA containing reduction-responsive modifications

Article abstract of DOI:10.1039/c6ob01272h

An original post-synthetic method on a solid support was developed to introduce various disulfide bond containing groups at the 2′-OH of oligoribonucleotides (RNAs). It is based on a thiol disulfide exchange reaction between several readily accessible alk

Full text of DOI:10.1039/c6ob01272h

Organic &
Biomolecular Chemistry
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A versatile post-synthetic method on a solid
support for the synthesis of RNA containing
reduction-responsive modifications†
Cite this: Org. Biomol. Chem., 2016,
14, 7010
Annabelle Biscans, Sonia Rouanet, Jean-Jacques Vasseur, Christelle Dupouy* and
Françoise Debart*
An original post-synthetic method on a solid support was developed to introduce various disulfide bond
containing groups at the 2’-OH of oligoribonucleotides (RNAs). It is based on a thiol disulfide exchange
reaction between several readily accessible alkyldisulfanyl-pyridine derivatives and 2’-O-acetylthiomethyl
RNA in the presence of butylamine. By this strategy, diverse 2’-O-alkyldithiomethyl RNAs were obtained.
These modifications provided high nuclease resistance to RNA and were easily removed with glutathione
treatment, thus featuring a potential use for siRNA prodrugs.
Received 13th June 2016,
Accepted 21st June 2016
DOI: 10.1039/c6ob01272h
www.rsc.org/obc
short interfering ribonucleic neutrals (siRNNs) whose inter-
nucleoside backbones containing S-acetyl-2-thioethyl (SATE)
Introduction
Small interfering RNAs (siRNAs) have attracted considerable phosphotriester groups9 are unmasked inside the cells by
attention because they play an important role in gene silencing thioesterases.10 The results obtained with the above described
strategies.1,2 However, their poor cell permeation and their low strategies are very encouraging and show that prodrug
resistance to nucleases remain the major hurdles to their approaches are promising for the development of siRNA thera-
development as therapeutics. To circumvent these problems, peutics.11 Among other possible transient modifications, bio-
chemists introduced several permanent chemical modifi- labile groups containing a disulfide link are likewise attractive
cations into siRNAs.3,4 Despite an increase of the enzymatic in prodrug strategies.12–15 Indeed, disulfide bonds are reduced
stability of siRNA, it is difficult to predict the influence of the to thiol groups under reducing conditions such as in the pres-
permanent modifications on siRNA activity which can signifi- ence of glutathione whose intracellular concentration ranges
cantly reduce the gene knockdown efficiency. Another way to from 1 mM to 10 mM whereas the extracellular concentration
overcome this drawback is to use temporary biolabile modifi- is low.16,17 Such biolabile modifications might also confer
cations which are designed to be removed inside cells to liber- nuclease resistance to siRNA and enhance their cellular
ate the active siRNA. Over the last few years, our research has uptake. Thus, we developed a facile chemical synthesis to
focused on a prodrug-based approach using 2′-O-acetalester introduce various alkyldithiomethyl (RSSM) groups containing
groups at 2′-OH of RNAs, which should be removed by carboxy- either lipophilic moieties or polar groups at 2′-OH of RNA
esterases. These transient modifications increased the siRNA (Fig. 1). These modifications were designed to be cleaved in a
stability towards enzymes, enhanced their cellular uptake and two-step process: (1) the reduction of the disulfide bond
promoted gene silencing after spontaneous naked delivery.5–8 leading to an unstable thiohemiformacetal, (2) the release of a
In the same way, recently Dowdy and colleagues evaluated
Department of Nucleic Acids, IBMM, UMR 5247, CNRS, Université de Montpellier,
ENSCM, UM Campus Triolet, Place E. Bataillon, 34095 Montpellier Cedex 05,
France. E-mail: christelle.dupouy@umontpellier.fr, françoise.debart@umontpellier.fr
†Electronic supplementary information (ESI) available: NMR spectra of uridine
phosphoramidite 1 and alkyldisulfanyl pyridine reagents, analytical data of
RNAs, Tm data and CD spectra of RNA duplexes, MALDI-TOF MS spectra with
SVPDE, and IEX-HPLC chromatograms with glutathione. See DOI:
10.1039/c6ob01272h
Fig. 1 Alkyldithiomethyl (RSSM) modifications at 2’-OH of RNA.
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thioformaldehyde generating the native RNA. As far as we
know, only a few examples of 2′-O-RSSM RNAs have been
reported. First, the tert-butyldithiomethyl (DTM) group was
described as a 2′-O-protecting group for RNA synthesis.18,19
However, due to the instability of the disulfide bond in a 2′-O-
DTM phosphoramidite derivative, because the phosphite triester
moiety can attack the disulfide bond intramolecularly, the use
of this protection was rather limited.20 To avoid this unwanted
reaction an elegant post-synthetic approach was proposed for
the synthesis of 2′-O-methyldithiomethyl (2′-O-MeSSM) oligo-
nucleotides which were obtained by the conversion in a solu-
tion of appropriate 2′-O-(2,4,6-trimethoxybenzylthiomethyl)
oligonucleotides upon treatment with dimethyl-(methylthio)-
sulfonium tetrafluoroborate.21 The weakness of this post-
synthetic approach is that exclusively the 2′-O-MeSSM modifi-
cation can be incorporated into RNA. We now report a
robust versatile post-synthetic method on a solid support
leading readily to diverse modified 2′-O-alkyldithiomethyl
(2′-O-RSSM)/2′-OH RNAs. Different RSSM modifications
were shown to increase RNA resistance to degradation by a
3′-exonuclease and to be efficiently converted into 2′-OH under
reducing conditions.
Table 1 Synthesis of RSSPy derivatives 2a–g
No.
R group
Solvent
Yield (%)
81
2a
MeOH/DMF
1/1
2b
2c
MeOH/DMF
1/1
68
77
MeOH
2d
2e
MeOH
MeOH
61
56
2f
MeOH/DMF
1/1
77
43
2g
MeOH
Results and discussion
Synthesis of 2′-O-RSSM oligonucleotides
To form the disulfide bond, our method consists of a thiol–di-
sulfide exchange between a thiolate anion and an alkyldisulfa-
nyl-pyridine (RSSPy) derivative. This thiol–disulfide exchange
is rapid and irreversible because the pyridinesulfenyl group is
a good leaving group.22,23 The thiolate anion was generated
under basic conditions from the deprotection of a 2′-O-acetyl-
thiomethyl (2′-O-AcSM) derivative previously studied in our
lab (Scheme 1).8 In the partially modified 2′-O-RSSM/2′-OH
RNAs, the 2′-OH resulted from the removal of 2′-O-pivaloyloxy-
methyl (2′-O-PivOM) groups under the standard ammonia
treatment.24 Therefore, the synthesis of 2′-O-RSSM/2′-OH
Scheme 2 Synthesis of 2’-O-AcSM uridine phosphoramidite unit 1.
Reagents and conditions: (a) (iPr)2NP(Cl)OCNE, DIEA, DCM, rt, 2.5 h,
90%.
modified RNAs required the use of commercially available protected amidite uridine 1 was isolated with high purity in
2′-O-PivOM phosphoramidites, 2′-O-AcSM uridine phosphor- very good yield (90%) after the 3′-phosphitylation of 2′-O-acetylthio-
amidite 1 and RSSPy derivatives (Table 1). The 2′-O-AcSM methyl-5′-O-(4,4′-dimethoxytrityl)-uridine (Scheme 2).8
Scheme 1 Synthetic strategy for 2’-O-RSSM/2’OH RNAs.
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The RSSPy derivatives were prepared by the reaction of of the elongation, thiol–disulfide exchange was performed by a
diverse commercially available thiols (RSH) with 2,2′-dithiodi- treatment of 2′-O-AcSM/2′-O-PivOM RNAs still “on support”
pyridine in methanol or a mixture of MeOH/DMF according to with RSSPy derivatives 2a–g (100 equivalents per modification)
a described procedure (Table 1).25,26
and an anhydrous solution of butylamine in THF for 15 min at
Thus, some RSSPy reagents bearing different chemical room temperature. During this reaction, the cyanoethyl groups
functionalities (aromatic (2a, 2b), aliphatic (2d), alcohol (2c), protecting the phosphates were also removed. Then, in a
carboxylic acid (2e), amine (2f) and amino acid (2g)) were second step, an aqueous concentrated ammonia treatment was
obtained with satisfactory yields (from 43 to 81%).
applied at 30 °C for 3 h to remove nucleobase protection and
The trifluoroacetyl group was used to protect the amino 2′-O-PivOM groups, and release 2′-O-RSSM/2′-OH RNAs 4a–g
function of RSSPy derivatives 2f and 2g to avoid side reactions from the solid support. Crude RNAs 4a–g were analyzed and
during the synthesis of 2′-O-RSSM/2′-OH RNAs. This protecting purified by IEX-HPLC and characterized by MALDI-TOF mass
group remained stable during the formation of the disulfide spectrometry (Table 2 and ESI, Fig. S9–S15†). They were
link and was removed under standard ammonia deprotection. efficiently obtained with satisfactory yields (45–65%) and high
No protection was needed for alcohol (2c) and carboxylic acid purities (92–98%) (Fig. 2 and Table 2).
(2e, 2g) functions.
Several 21-mer RNAs 4a–g (Table 2) corresponding to the
sense strand of a siRNA targeting ICAM-1 mRNA with four
RSSM groups spread out over the sequence were synthesized
on an automated DNA synthesizer with uridine amidite 1 and
2′-O-PivOM phosphoramidites by using commercially available
controlled-pore glass (LCAA-CPG) linked to 5′-O-DMTr-dT
through a 3′-O-succinyl linker. The elongation of the RNAs was
carried out on a 1 µmol scale, following a published auto-
mated RNA synthetic procedure with a 180 s coupling step and
Fig. 2 IEX-HPLC and MALDI-TOF spectra of (A) crude and (B) purified
5-benzylmercaptotetrazole (BMT) as the activator.24 At the end RNA 4b.
Table 2 Data of 2’-O-RSSM/2’-OH RNAs
Sequence 5′ → 3′ a GCCURSSMCAGCACGURSSMACCURSSMCURSSMAdTdTb
MALDI-TOF MSd
RNA of
Purity of
Time of RNA
RNA
3h
R group
None
interestc (%)
Calcd
Found
RNAe (%)
Tm f (°C)
81.7
ΔTm g (°C)
—
degradationi (min)
71
65
6564.73
7236.58
6563.16
7235.76
100
7
4a
92
79.7
−2.0
>20
4b
4c
62
61
7292.02
7044.15
7290.38
7044.07
92
95
79.7
80.7
−2.0
−1.0
>20
15
4d
4e
59
64
6988.22
7164.59
6987.87
7163.64
95
97
79.7
79.7
−2.0
−2.0
20
15
4f
45
58
7048.52
7224.41
7048.36
7224.00
98
98
80.7
80.7
−1.0
−1.0
>20
>20
4g
a URSSM = 2′-O-RSSM uridine; G, C, A, U = 2′-OH nucleosides. b Sense strand of siRNA targeting ICAM-1 mRNA. c Percentages of RNA of interest
present in the crude samples after RNA deprotection determined by IEX-HPLC. d Negative mode. e Purity of oligonucleotides after purification by
IEX-HPLC. f Tm values obtained from UV melting curves at 260 nm with 3 µM strand concentration in 10 mM sodium cacodylate, 100 mM NaCl,
pH 7.0. Data are the average of three hybridization-melting cycles. Estimated error in Tm
= 0.5 °C. Complementary RNA strand: UAGAGGUAC-
GUGCUGAGGCdTdT. g ΔTm is the difference in Tm relative to the 2′-OH unmodified duplex. h Unmodified sense strand of siRNA targeting ICAM-1
mRNA. i Nuclease stability of RNAs towards SVPDE determined by MALDI-TOF Mass Spectrometry. Time was determined when the main mass
peak corresponded to a 4-mer.
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Thermal stabilities
The influence of the different RSSM modifications on the
thermal stability of duplexes formed between RNAs 4a–g and
their complementary unmodified strand was evaluated by UV-
melting experiments at 260 nm (Table 2 and ESI, Fig. S16†).
Compared to the unmodified duplex with RNA 3 (Tm
=
81.7 °C), all duplexes containing 2′-O-RSSM groups were
slightly destabilized (ΔTm = −1.0 °C to −2.0 °C). Nevertheless,
these destabilizations (ΔTm ≤ −0.5 °C per modification) were
not detrimental to the formation of stable duplexes under
physiological conditions. In addition, circular dichroism (CD)
spectra of all modified duplexes indicated the typical curve of
an A-form helix geometry with a positive band near 264 nm
and a strong negative band at 210 nm (ESI, Fig. S17†). The
shapes of the CD spectra were similar to that of the unmodi-
fied duplex. Therefore, whatever the RSSM modification the
original A-form conformation of the RNA duplex was
maintained.
Fig. 3 IEX-HPLC analysis of the reductive conversion of the duplex
with RNA 4a into the unmodified duplex with RNA 3 by treatment with
5.6 mM glutathione in 7.5 mM HEPES buffer (pH 8) for 1 h.
After 1 h incubation, as shown in Fig. 3 for the duplex with
RNA 4a, all the peaks corresponding to the disulfide-contain-
ing duplexes have disappeared and the native 2′-OH duplex
was mainly generated (with 70 to 95% purity). A shoulder peak
corresponding to the thiohemiformacetal intermediate was
also noticeable in the chromatograms for all RSSM groups
(ESI, Fig. S20 and Table S1†). The disulfide bond reduction
has proceeded rapidly and cleanly whereas the complete con-
version into 2′-OH RNA has required a longer time under these
experimental conditions.
Nuclease resistance
The stability of 2′-O-RSSM/2′-OH RNAs 4a–g towards a 3′-exo-
nuclease (snake venom phosphodiesterase (SVPDE)) was evalu-
ated by MALDI-TOF mass spectroscopy (Table 2 and ESI,
Fig. S18†).27 All the 2′-O-RSSM/2′-OH RNAs 4a–g exhibited
resistance to SVPDE compared to unmodified RNA 3 which
was degraded within 7 min. It is noteworthy that a correlation
between the RNA degradation in the presence of SVPDE and
the nature of the RSSM group can be established. Indeed, the
more lipophilic the RSSM group, the more stable the RNA
towards SVPDE.28 At 20 min, RNAs bearing alcohol (4c), car-
boxylic acid (4e) and aliphatic moieties (4d) were nearly totally
degraded whereas for RNAs with aromatic groups (4a and 4b)
a peak corresponding to a 10-mer was still noticeable in the
mass spectrum (ESI, Fig. S18†). Moreover, RNAs with RSSM
containing amine groups (4f and 4g), protonated at physiologi-
cal pH, had a similar stability to RNAs bearing aromatic
groups (4a and 4b) (Table 2). This relative stability could be
due to the presence of positive charges, which induce unfavor-
able interactions with the SVPDE metallic ion essential for the
enzyme activity.29,30
This preliminary study performed under physiological con-
ditions anticipates that all 2′-O-RSSM groups should be
efficiently reduced within the cells releasing the unmodified
2′-OH duplex.
Conclusions
We developed an original and straightforward post-synthetic
method on a solid support, to introduce efficiently a large
variety of alkyldithiomethyl groups at the 2′-OH of RNA. This
method is versatile since from one 2′-O-AcSM-containing RNA,
we can obtain numerous 2′-O-RSSM-modified RNAs bearing
lipophilic or polar groups depending on the alkyldisulfanyl-
pyridine derivative used for the disulfide–thiol exchange. The
Glutathione treatment
To use 2′-O-RSSM/2′-OH modified siRNAs in
a prodrug results demonstrated that RSSM modifications did not disrupt
approach, the disulfide bond of 2′-O-RSSM groups should be the duplex stability dramatically while maintaining an A-form
converted into 2′-OH in an intracellular reductive environment. conformation and they provided RNA resistance against 3′-exo-
First, the reductive conversion of 2′-O-RSSM RNA 4a into the nuclease. These features are promising for designing effective
corresponding 2′-OH RNA 3 was evaluated in 5.6 mM gluta- siRNA constructs. In addition, an efficient and rapid conver-
thione in 7.5 mM HEPES buffer (pH 8) and monitored by sion of 2′-O-RSSM groups into 2′-OH under reducing con-
IEX-HPLC. After 1 h incubation, in the HPLC chromatogram, ditions similar to those found in the intracellular medium
the new major peak was assigned to the 2′-OH RNA 3 but a shows the great potential of 2′-O-RSSM/2′-OH RNAs for their
shoulder peak was also noticeable. The MALDI-TOF mass ana- use as prodrugs of siRNA. To extend the scope of applications
lysis showed that this peak corresponds to the hemithioacetal of 2′-O-RSSM RNAs and for the design of potent siRNAs, the
species resulting from the disulfide bond cleavage and the synthesis of other 2′-O-AcSM phosphoramidite ribonucleotides
subsequent incomplete degradation of the unstable intermedi- is in progress. We anticipate that this post-synthetic method
ate within 1 h of incubation (ESI, Fig. S19†). The same experi- would be also attractive in the context of RNA cross-linking with
ment was conducted with 2′-O-RSSM duplexes with RNAs 4a–g. other biomolecules (nucleic acids, peptides, proteins, sugars).
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ethyl acetate (80 mL) and washed with saturated aqueous
NaHCO3 (3 × 40 mL) and brine (40 mL). The organic layer was
dried over Na2SO4, filtered and concentrated under reduced
Experimental section
General
CH3CN, MeOH, pyridine and DIEA were distilled over calcium pressure. The crude material was purified by silica gel column
hydride. All reactions were performed under anhydrous con- chromatography with a mixture of cyclohexane and ethyl
ditions under argon. NMR experiments were accomplished on acetate (80 : 20). The desired compound 2a was obtained as
Bruker DRX 400 and AM 300 spectrometers at 20 °C. HRMS clear oil (759 mg, 3.25 mmol, 81%). 1H-NMR (300 MHz,
analyses were performed with electrospray ionization (ESI) in CDCl3) δ 8.43 (dq, J = 4.8 Hz, J = 2.4 Hz, 1H, Hpy); 7.54–7.47
positive or negative mode on a Q-TOF Micromass spectro- (m, 2H, Hpy); 7.32–7.20 (m, 5H, Ar); 7.09 (ddd, J = 6.3 Hz, J =
meter. Analytical and semi-preparative HPLC were performed 4.8 Hz, J = 2.4 Hz, 1H, Hpy); 4.02 (s, 2H, CH2). 13C-NMR
on a Dionex DX 600 HPLC system equipped with anion- (100 MHz, CDCl3) δ 160.0 (Cq); 149.3 (Cpy); 136.9 (Cpy); 136.5
exchange DNAPac PA 100 columns (4 × 250 mm for analysis or (Cq); 129.3, 128.5, 127.6 (CAr); 120.5 (Cpy); 119.6 (Cpy); 43.7
9 × 250 mm, Dionex). The following HPLC solvent systems (CH2). HRMS (ESI+) m/z calcd for C12H11NS2 (M + H)+
were used: 20% CH3CN in 25 mM Tris-HCl buffer, pH 8 (buffer 234.0411, found 234.0410
A) and 20% CH3CN containing 200 mM NaClO4 in 25 mM
Tris-HCl buffer, pH 8 (buffer B). Flow rates were 1.5 mL min−1 2-(Phenylethyldisulfanyl)pyridine (2b)
and 5 mL min−1 for analysis and semi-preparative purposes,
respectively. MALDI-TOF mass spectra were recorded on a
Using the same procedure as for the synthesis of 2a, starting
from phenylethylmercaptan (0.50 g, 3.62 mmol) compound 2b
Voyager-DE spectrometer equipped with a N2 laser (337 nm)
was obtained as yellow oil (608 mg, 2.46 mmol, 68%). 1H-NMR
(Perseptive Biosystems, USA) using 2,4,6-trihydroxyaceto-
(300 MHz, CDCl3) δ 8.48 (dq, J = 4.8 Hz, J = 1.2 Hz, 1H, Hpy);
7.70–7.59 (m, 2H, Hpy); 7.32–7.17 (m, 5H, Ar); 7.09 (ddd, J =
phenone as a saturated solution in a mixture of acetonitrile/0.1 M
ammonium citrate solution (1 : 1, v/v) for the matrix. Analytical
samples were mixed with the matrix in a 1 : 5 (v/v) ratio, crystal-
lized on a 100-well stainless steel plate and analyzed. UV quan-
titation of RNAs was performed on a Varian Cary 300 Bio
UV/Visible spectrometer by measuring absorbance at 260 nm.
(M + H)+ 248.0568, found 248.0571.
6.9 Hz, J = 4.8 Hz, J = 1.2 Hz, 1H, Hpy); 3.08–2.99 (m, 4H, CH2).
13C-NMR (75 MHz, CDCl3) δ 160.3 (Cq); 149.5 (Cpy); 139.6 (Cq);
137.2 (Cpy); 128.6, 128.5, 126.5 (Carom); 120.6 (Cpy); 119.7 (Cpy);
40.0 (CH2); 35.3 (CH2). HRMS (ESI+) m/z calcd for C13H13NS2
2′-O-Acetylthiomethyl-3′-O-((2-cyanoethyl)(diisopropylamino)-
phosphanyl)-5′-O-(4,4′-dimethoxytrityl) uridine (1)
2-(Pyridin-2-yldisulfanyl)ethan-1-ol (2c)
To
a stirred solution of 2,2′-dithiodipyridine (2.11 g,
To a solution of 2′-O-acetylthiomethyl-5′-O-(4,4′-dimethoxytri-
tyl) uridine (4.28 g, 6.74 mmol, 1.00 equiv.) in anhydrous
CH2Cl2 (52 mL) previously passed through an alumina column
was added dropwise a mixture of N,N-diisopropylethylamine
(2.94 mL, 16.85 mmol, 2.50 equiv.) and 2-cyanoethyl N,N-di-
isopropylchlorophosphoramidite (3.31 mL, 14.83 mmol, 2.20
equiv.) in CH2Cl2. The mixture was stirred for 2.5 h at room
temperature under argon. After reaction completion, ethyl
acetate previously washed with a saturated aqueous NaHCO3
solution was added and the reaction mixture was poured into
a saturated NaCl/NaHCO3 solution (1/1 v/v). The aqueous layer
was extracted with ethyl acetate and organic layers were dried
over Na2SO4. The solvent was concentrated under reduced
pressure. The crude material was purified by silica gel column
chromatography with an isocratic elution of CH2Cl2 and
acetone (9/1) containing 1% pyridine. The desired phosphor-
amidite 1 was obtained as white foam (5.05 g, 6.05 mmol, 90%).
31P-NMR (121 MHz, CD3CN): δ 150.0, 149.3. HRMS (ESI+) m/z
calcd for C42H51N4O10PS (M + H)+ 835.3142, found 835.3149.
9.60 mmol, 1.50 equiv.) in MeOH (50 mL), was added dropwise
a solution of 2-mercaptoethanol (0.50 g, 6.40 mmol, 1.00
equiv.) in MeOH (12 mL). The yellow mixture was stirred under
an argon atmosphere at room temperature for 30 minutes. The
mixture was concentrated under reduced pressure. The crude
material was purified by silica gel column chromatography
with a mixture of cyclohexane and ethyl acetate (60 : 40). The
desired compound 2c was obtained as a yellow solid (923 mg,
4.94 mmol, 77%). 1H-NMR (400 MHz, CDCl3) δ 8.47 (d, J =
4.8 Hz, 1H, Hpy); 7.56 (t, J = 8.0 Hz, 1H, Hpy); 7.39 (d, J =
8.4 Hz, 1H, Hpy); 7.12 (dd, J = 7.2 Hz, J = 5.2 Hz, 1H, Hpy); 3.78
(t, J = 5.2 Hz, 2H, CH2); 2.93 (t, J = 5.2 Hz, 2H, CH2). 13C-NMR
(100 MHz, CDCl3) δ 159.0 (Cq); 149.7 (Cpy); 136.8 (Cpy); 121.8
(Cpy); 121.4 (Cpy); 58.2 (CH2); 42.6 (CH2). HRMS (ESI+) m/z
calcd for C7H9NOS2 (M + H)+ 188.0204, found 188.0205.
Ethyldisulfanyl-pyridine (2d)
Using the same procedure as for the synthesis of 2c, starting
from ethanethiol (0.50 g, 8.05 mmol) compound 2d was
obtained as yellow oil (835 mg, 4.88 mmol, 61%). 1H-NMR
2-(Benzyldisulfanyl)pyridine (2a)
To
a stirred solution of 2,2′-dithiodipyridine (1.33 g, (400 MHz, CDCl3) δ 8.42 (dq, J = 4.8 Hz, J = 0.8 Hz, 1H, Hpy);
6.04 mmol, 1.50 equiv.) in 1 : 1 MeOH/DMF (50 mL), was 7.69 (dt, J = 8.0 Hz, J = 0.8 Hz, 1H, Hpy); 7.60 (td, J = 8.0 Hz, J =
added dropwise a solution of benzyl mercaptan (0.50 g, 2.0 Hz, 1H, Hpy); 7.04 (ddd, J = 7.6 Hz, J = 4.8 Hz, J = 0.8 Hz,
4.03 mmol, 1.00 equiv.) in 1 : 1 MeOH/DMF (8 mL). The yellow 1H, Hpy); 2.77 (q, J = 7.6 Hz, 2H, CH2); 1.30 (t, J = 7.6 Hz, 3H,
mixture was stirred under an argon atmosphere at room temp- CH3). 13C-NMR (100 MHz, CDCl3) δ 160.5 (Cq); 149.4 (Cpy);
erature for 30 minutes. Then the mixture was diluted with 136.9 (Cpy); 120.4(Cpy); 119.4 (Cpy); 32.7 (CH2); 14.1 (CH3).
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HRMS (ESI+) m/z calcd for C7H9NS2 (M + H)+ 172.0255, found obtained as a pale brown solid (287 mg, 0.88 mmol, 43%).
172.0257.
1H-NMR (400 MHz, DMSO-d6) δ 9.90 (d, J = 7.6 Hz, 1H, NH);
8.46 (dq, J = 4.8 Hz, J = 1.2 Hz, 1H, Hpy); 7.79 (td, J = 8.0 Hz, J =
1.6 Hz, 1H, Hpy); 7.71 (dt, J = 8.0 Hz, J = 1.2 Hz, 1H, Hpy); 7.26
3-(Pyridin-2-yldisulfanyl)propionic acid (2e)
Using the same procedure as for the synthesis of 2c, starting (ddd, J = 7.6 Hz, J = 4.8 Hz, J = 1.2 Hz, 1H, Hpy); 4.55 (m, 1H,
from 3-mercaptopropionic acid (0.50 g, 4.71 mmol) compound CH); 3.35 (dd, J = 14.0 Hz, J = 4.0 Hz, 1H, CH2); 3.21 (dd, J =
2e was obtained as a yellow solid (570 mg, 2.65 mmol, 56%). 14.0 Hz, J = 10.4 Hz, 1H, CH2). 13C-NMR (100 MHz, DMSO-d6)
1H-NMR (400 MHz, DMSO-d6) δ 8.45 (dq, J = 4.8 Hz, J = 1.2 Hz, δ 170.1 (CvO); 158.2 (Cq); 156.5 (q, CvO); 149.7 (Cpy); 137.7
1H, Hpy); 7.81 (td, J = 8.0 Hz, J = 1.6 Hz, 1H, Hpy); 7.75 (dt, J = (Cpy); 121.4 (Cpy); 119.5 (Cpy); 117.0 (q, CF3); 51.9 (CH); 38.6
8.0 Hz, J = 1.2 Hz, 1H, Hpy); 7.23 (ddd, J = 7.2 Hz, J = 4.8 Hz, J = (CH2). 19F-NMR (160 MHz, DMSO-d6) δ −74.32. HRMS (ESI+)
1.2 Hz, 1H, Hpy); 3.00 (t, J = 6.8 Hz, 2H, CH2); 2.64 (t, J = m/z calcd for C10H9N2O3F3S2 (M + H)+ 327.0085, Found
6.8 Hz, 2H, CH2). 13C-NMR (100 MHz, DMSO-d6) δ 172.5 327.0086.
(CvO); 159.0 (Cq); 149.6 (Cpy); 137.8 (Cpy); 121.2 (Cpy); 119.3
Synthesis of partially modified 2′-O-AcSM oligonucleotides
(Cpy); 33.4 (CH2); 33.3 (CH2). HRMS (ESI+) m/z calcd for
C8H9NO2S2 (M − H)− 213.9996, found 213.9997.
Oligonucleotides were synthesized on an ABI model 394
DNA/RNA synthesizer on a 1 µmol scale using commercially
available 2′-O-PivOM phosphoramidites (Chemgenes), 2′-O-
2,2,2,-Trifluoro-N-(2-(pyridin-2-yldisulfanyl)ethyl)acetamide (2f)
To a stirred solution of cysteamine (0.50 g, 4.40 mmol, AcSM uridine phosphoramidite 1 and a controlled-pore glass
1.00 equiv.) in methanol (3 mL) under argon, was added tri- (CPG) commercial solid support. Oligonucleotides were
ethylamine (594 µL, 4.40 mmol, 1.00 equiv.) and ethyl assembled in TWIST™ synthesis columns (Glen Research).
trifluoroacetate (597 µL, 5.50 mmol, 1.25 equiv.). The mixture Phosphoramidites were vacuum dried prior to their dis-
was stirred at room temperature for 3.5 h and then evaporated solution in extra dry acetonitrile (Biosolve) at 0.1 M con-
under pressure. The residue was dissolved in 1 : 1 MeOH/DMF centration. Coupling for 180
s
was performed with
(8 mL) and solution of 2,2′-dithiodipyridine (1.46 g, 5-benzylmercaptotetrazole (BMT, 0.3 M) as an activator. The
a
6.60 mmol, 1.50 equiv.) in 1 : 1 MeOH/DMF (50 mL) was added oxidizing solution was 0.1 M iodine in THF/pyridine/H2O
dropwise. The yellow mixture was stirred under an argon atmo- (78 : 20 : 2; v/v/v) (Link Technologies). The capping step was
sphere at room temperature for 2 h. Then the mixture was performed with a mixture of 5% phenoxyacetic anhydride
diluted with ethyl acetate (80 mL) and washed with saturated (Pac2O) in THF and 10% N-methylimidazole in THF (Link
aqueous NaHCO3 (3 × 40 mL) and brine (40 mL). The organic Technologies). Detritylation was performed with 3% TCA in
layer was dried over Na2SO4, filtered and concentrated under CH2Cl2. After RNA assembly completion, the column was
reduced pressure. The crude material was purified by silica gel removed from the synthesizer and dried under a stream
column chromatography with dichloromethane. The desired of argon.
compound 2f was obtained as a yellow solid (951 mg,
Synthesis of partially modified 2′-O-RSSM oligonucleotides (4a–g)
3.37 mmol, 77%). 1H-NMR (400 MHz, CDCl3) δ 9.61 (s, 1H,
NH); 8.47 (d, J = 4.4 Hz, 1H, Hpy); 7.60 (td, J = 8.0 Hz, J = 1 mL of a 0.4 M alkyldisulfanyl-pyridine 2a–g (400 equiv.) solu-
1.2 Hz, 1H, Hpy); 7.40 (dt, J = 8.0 Hz, J = 1.2 Hz, 1H, Hpy); 7.19 tion and a 2.5 M butylamine (250 µL) solution in anhydrous
(ddd, J = 8.0 Hz, J = 4.4 Hz, J = 1.2 Hz, 1H, Hpy); 3.67 (q, J = THF (750 µL) was applied to the column containing the
6.0 Hz, 2H, CH2); 2.93 (m, 2H, CH2). 13C-NMR (100 MHz, 2′-O-AcSM oligonucleotide for 15 min using two glass syringes
CDCl3) δ 158.6 (Cq); 157.3 (q, CvO); 149.7 (Cpy); 137.0 (Cpy); filled with 4 Å molecular sieves (5 beads each). Then the solu-
122.3 (Cpy); 121.9 (Cpy); 117.0 (q, CF3); 37.9 (CH2); 36.6 (CH2). tion was removed and the solid-support was washed with an-
19F-NMR (160 MHz, CDCl3) δ −75.91. HRMS (ESI+) m/z calcd hydrous THF followed by a 1 min flush with argon. The solid
for C9H9N2OF3S2 (M + H)+ 283.0187, found 283.0189.
support was treated with a 30% aqueous ammonia solution for
3 h. The deprotection solution was evaporated in the presence
of isopropylamine (13% of total volume) under reduced
S-(Pyridin-2-ylthio)-N-(2,2,2-trifluoroacetyl)cysteine (2g)
To a stirred solution of L-cysteine (250 mg, 2.06 mmol, 1.00 pressure. The crude oligonucleotide was diluted in water,
equiv.) in methanol (2 mL) under argon were added triethyl- transferred in a 2 mL Eppendorf vial and then lyophilized
amine (278 µL, 2.06 mmol, 1.00 equiv.) and ethyl trifluoro- from water.
acetate (280 µL, 2.57 mmol, 1.25 equiv.). The mixture was stirred
at room temperature overnight and then evaporated under
pressure. The residue was dissolved in MeOH (4 mL) and a
Analysis, purification and desalting of 2′-O-RSSM
oligonucleotides (4a–g)
solution of 2,2′-dithiodipyridine (520 mg, 2.37 mmol, 1.15 The crude 2′-O-RSSM oligonucleotides 4a–g were analyzed by
equiv.) in MeOH (12 mL) was added dropwise. After stirring anion-exchange HPLC using a specific linear gradient of buffer
for 30 minutes, the yellow mixture was evaporated under B in buffer A for each RNA, and they were characterized by
reduced pressure. The crude material was purified by silica gel MALDI-TOF spectrometry. The crude mixtures were then puri-
column chromatography with a step gradient of dichloro- fied by semi-preparative IEX-HPLC with a specific linear gradi-
methane and methanol (5–8%). The desired compound 2g was ent of buffer B in buffer A. The pure fractions of RNA were
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pooled in a 100 mL round-bottomed flask and were concen- 5.6 mM). After 1 h incubation at 37 °C, the samples were ana-
trated to dryness under reduced pressure. The residues were lyzed by IEX-HPLC and MALDI-TOF MS.
dissolved in 100 mM TEAAc buffer, pH 7 (8 mL divided into
three portions for rinsing the flask: 5 mL, 2 mL, and 1 mL)
and were loaded on a C18 cartridge (Waters, Sep-Pak®). Elution
was performed with 10 mL of a 100 mM TEAAc solution then
Acknowledgements
with a 10 mL of 50% CH3CN in 12.5 mM TEAAc solution. The Annabelle Biscans thanks the University of Montpellier for
second fraction containing the desired compound was col- financial support.
lected in a 100 mL round-bottomed flask and was freeze-dried.
The residue was dissolved in 1.5 mL water (divided into 3 por-
tions of 0.8 mL, 0.4 mL, and 0.3 mL for rinsing the flask) and
transferred to a 2 mL Eppendorf-vial and lyophilized from
Notes and references
water. Purified oligonucleotides 4a–g were analyzed by MALDI-
TOF analysis and quantified by UV measurement. Lyophilized
RNAs were stored at −20 °C for several months without any
degradation.
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Org. Biomol. Chem., 2016, 14, 7010–7017 | 7017