Synthesis of Adenine Dinucleosides 2′,5′-Bridged by Sulfur-Containing Linkers as Bisubstrate SAM Analogues for Viral RNA 2′-O-Methyltransferases

Article abstract of DOI:10.1002/ejoc.201901120

Viral RNA 2′-O-methyltransferases play a crucial role for luring the host cell innate antiviral response during a viral infection by catalyzing either the methylation of the 5′-end RNA cap-structure at 2′-OH of nucleoside N1 or by inducing internal 2′-O-methylation of adenosines within RNA sequence using S-adenosyl-l-methionine (SAM) as the methyl donor. Our goal is to synthesize bisubstrate SAM analogues mimicking the transition state of the 2′-O-methylation of the RNA in order to block viral 2′-O-methyltransferases and struggle against emerging viruses. Here we designed and synthesized five dinucleosides by connecting a 5′-thioadenosine representing the SAM to the 2′-OH of another adenosine unit mimicking the RNA substrate, via various sized sulfur-containing linkers such as alkylthioether linkers, sulfoxide or sulfone derivatives, or a disulfide bond.

Full text of DOI:10.1002/ejoc.201901120

DOI: 10.1002/ejoc.201901120
Full Paper
RNA Methyltransferases | Very Important Paper |
Synthesis of Adenine Dinucleosides 2′,5′-Bridged by Sulfur-
Containing Linkers as Bisubstrate SAM Analogues for Viral RNA
2′-O-Methyltransferases
Rostom Ahmed-Belkacem,[a] Priscila Sutto Ortiz,[b] Etienne Decroly,[b] Jean-Jacques Vasseur,[a]
and Françoise Debart*[a]
Abstract: Viral RNA 2′-O-methyltransferases play a crucial role tion state of the 2′-O-methylation of the RNA in order to block
for luring the host cell innate antiviral response during a viral viral 2′-O-methyltransferases and struggle against emerging
infection by catalyzing either the methylation of the 5′-end RNA viruses. Here we designed and synthesized five dinucleosides
cap-structure at 2′-OH of nucleoside N1 or by inducing internal by connecting a 5′-thioadenosine representing the SAM to the
2′-O-methylation of adenosines within RNA sequence using S- 2′-OH of another adenosine unit mimicking the RNA substrate,
adenosyl-L-methionine (SAM) as the methyl donor. Our goal is via various sized sulfur-containing linkers such as alkylthioether
to synthesize bisubstrate SAM analogues mimicking the transi- linkers, sulfoxide or sulfone derivatives, or a disulfide bond.
Introduction
ger RNAs, protects viral RNA from degradation by cellular nucle-
ases. Particularly, our research aims at studying and targeting
Emerging RNA viruses (e.g., Dengue, Zika, SARS, MERS, Ebola viral RNA Methyltransferases (MTases) which play a crucial role
viruses) are important human pathogens causing substantial by catalyzing the methylation of the RNA cap-structure using
health and economic burden.[1] Their spreading is, among oth- S-adenosyl-
L-methionine (SAM) as the methyl donor. Viral N7-
ers, linked to their rapid evolution combined with their capacity MTase methylates the cap at the nitrogen in position N7 of
to escape antiviral response by hiding their RNA from detection guanosine in order to allow RNA translation into viral pro-
by antiviral sensors or restriction factors.[2] The viral replication/ teins.[3] The cap structure is also often methylated at the
transcription complex contains enzymes essential for virus repli- 2′-O-position of the N1 residue (adenosine or guanosine) by
cation, which are involved in RNA synthesis (polymerase) and 2′-O-MTase. Moreover, internal 2′-O-methylations of viral RNA
RNA capping. The cap structure, consisting of a guanosine have been demonstrated with Sudan ebolavirus, Dengue and
linked by a 5′-5′-triphosphate bridge to the 5′-end of messen- Zika viruses, and HIV.[4] These 2′-O- methylations were recently
Scheme 1. The 2′-O-methyltransferase reaction on the cap structure of a mRNA with its transition state intermediate.
evidenced as self-markers, hiding viral RNA from detection by
RIG-like receptors[2c,5] and limiting the restriction of viral replica-
tion by IFIT1/3 molecules.[6] It is now currently admitted that
these key enzymes are potent antiviral targets, as their inhibi-
tion will both unmask the viral RNA to the innate immunity and
limit the virus replication. Small-molecule RNA MTase inhibitors
[a] IBMM, UMR 5247, CNRS, University of Montpellier,
ENSCM, Montpellier, France
E-mail: francoise.debart@umontpellier.fr
[b] AFMB, CNRS, Aix-Marseille University,
UMR 7257, 163 avenue de Luminy, Marseille, France
Supporting information and ORCID(s) from the author(s) for this article are
available on the WWW under https://doi.org/10.1002/ejoc.201901120.
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Figure 1. Rationale for designing a library of diverse bisubstrate SAM analogues for RNA 2′-O-methyltransferases.
(Sinefungin, 5′-methylthioadenosine (MTA), SAM or S-adenosyl- MTases, SAM-adenosine conjugates mimicking the transition
-homocysteine (SAH) have already been described but these state of methylation at N6 were synthesized by connecting a
L
SAM analogs show inadequate selectivity due to the high ho- SAM analogue to the N6-position of an adenosine unit via alkyl
mology of SAM binding domain of the different RNA MTases.[7] and urea linkers.[14] The binding of these bisubstrate analogues
To overcome this lack of selectivity, we propose to develop for Ribosomal RNA large subunit MTase J (RlmJ) has been stud-
another approach with bisubstrate nucleosidic analogues as ied and they were shown to be useful as starting scaffolds for
2′-O-MTase inhibitors by mimicking the transition state of the inhibitor design against m6A RNA MTases.[15] Beside these few
2′-O-methylation of the RNA cap structure (Scheme 1).[7]
These analogues consist of a SAM analogue without the
amino acid side chain, covalently bound to the 2′-OH of an
adenosine unit via various sized linkers containing one or two
sulfur atoms. In this bisubstrate approach, the SAM analogue
has been designed to accommodate the SAM binding pocket
of the 2′-O-MTase and the adenosine unit represents the 5′-
end nucleoside (N1) of mRNA to fit in the RNA binding pocket
(Figure 1).
examples, none other RNA MTases have been targeted by bi-
substrate analogues.
In this work, we report on the synthesis of five S-adenosyl-
5′-thioadenosine conjugates as bisubstrate analogues for the
study of RNA 2′-O-methyltransferases. All these compounds
were designed with a 5′-thioadenosine linked to the 2′-OH of
an adenosine unit through alkyl linkers of various length
(methyl or ethyl) and/or different oxidation degrees of the
sulfur atom. Indeed, we first focused on the sulfide-containing
In the same way, several bisubstrates for diverse methyltrans- linkers (S-linkers) that represents the most fitting motives to
ferases (DNA MTases,[8] catechol MTase,[9] protein MTases,[10] mimic the SAM structure closely in comparison to other linka-
nicotinamide MTase[11]) have been previously reported in the ges with diverse heteroatoms in place of S. Moreover, S-linkers
literature. Nevertheless, it is noteworthy some relevant exam- are attractive as they are stable and non-hydrolysable. This was
ples of the use of bisubstrate nucleosidic analogues for the previously shown with the synthesis of several 2′-dialkyl S-
study of nucleic acids methyltransferases. Particularly, Arimondo linked dinucleosides which were incorporated into oligonucleo-
developed transition state analogues of DNA methylation based tide analogues for the study of their hybridization properties in
on the coupling of cytosine analogues to adenosine to give antisense purposes.[16] Furthermore, the corresponding sulfox-
5-methylcytosine-adenosine compounds.[12] Moreover, the first ides and sulfones are of interest for chemists as they result from
bisubstrates targeting RNA methyltransferases have been de- simple thioether oxidation and these groups are commonly
scribed in 1986 and one compound designed with the SAM found in nature as well as in the structure of some active drugs
moiety linked to the C6 of a guanine derivative demonstrated (Disulone®, Modiodal®). The presence of sulfoxide and sulfone
an inhibitory activity against vaccinia RNA N7-guanine MTase was also noted in aminoglycoside-Coenzyme A bisubstrates tar-
for the N7-methylation of the 5′-cap structure.[13] More recently, geting aminoglycoside N-6′-acetyltransferase.[17] Recently, two
in the context of deciphering the roles of N6m-A RNA modifica- SAM structural analogues, a sulfoxide and a sulfone derived
tions and consequently exploring the functions of N6-A RNA from SAH have been synthesized as substrates for the study of
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their reductive cleavage by radical SAM enzymes.[18] In addition, ously described by our group,[21] bearing a pro-nucleophile site
it should be mentioned that a cytosine or a thymine dinucleo- at 2′-position, and the commercially available 5′-O-tosyl adeno-
side 3′,5′-bridged by a sulfone-containing linker has been de- sine or 5′-Cl-adenosine, both bearing an electrophile site at
scribed in the literature for the synthesis of stable sulfonyl-con-
taining antisense oligonucleotides.[19] Inspired by this work, the
S-linked dinucleoside 1 has been oxidized in sulfoxide (SO, 2)
or sulfone (SO2, 3) since these sulfur functional groups might
improve chemical stability and solubility in water of the bisub-
strates (Figure 1). Likewise, the disulfide bridge represents an
attractive functional group to design another S-linker. The di-
sulfide bonds are widely found in natural biological systems
and play a central role in protein stability, they are able to un-
dergo disulfide-exchange reactions with thiols over a broad
range of pH. Interestingly, some disulfide dinucleosides as
nicotinamide adenine dinucleotide (NAD) mimics were reported
to inhibit several NAD kinases.[20] In similar way, we linked two
adenosines via an alkyl disulfide bond to yield the 2′,5′-di-
sulfanyl dinucleoside 5.
5′-position. A basic medium (7 M NH3/MeOH or BuNH2/THF)
released a nucleophilic thiolate at 2′-position of A1 prone to
react with A2. Nevertheless, the dinucleoside coupling was un-
successful due to the too fast degradation of the 2′-thiohemi-
acetal species into adenosine. In the second strategy, the reac-
tivity centers were reversed in the synthons A1 and A2 with a
thioacetyl group as the pro-nucleophile site at 5′-position of A2
and a 2′-chloromethyl group as electrophile in A1. The coupling
was similarly achieved in basic medium to generate the nucleo-
philic thiolate in A2, capable to attack the chloromethyl group.
Following this strategy, we first synthesized the 2′-chloro-
methyl derivative 6 from the commercial 3′,5′-O-tetraisoprop-
yldisiloxane (TIPDS) N6-phenoxyacetyl adenosine via a 2′-O-
methylthiomethyl derivative (Pummerer rearrangement) upon
a described procedure (Scheme 2).[21] This compound 6 remains
stable for 2 h at room temperature therefore requires a rapid
utilization in the next coupling with a 5′-thiolate. In parallel,
the introduction of the thioacetyl group at 5′-position of 2′,3′-
isopropylideneadenosine was achieved by a Mitsunobu reac-
tion with 98 % yield to give compound 7 which was subse-
quently 2′,3′-deprotected in acidic conditions affording 5′-thio-
Results and Discussion
Synthesis of adenine dinucleoside 1 with a methylthioether
linker. Two strategies have been tested to obtain the dinucleo-
side 1 with a methylthioether linker between both adenosines
A1 and A2 (Figure 1). The first one has consisted in the coupling acetyl adenosine 8.[22] Then, several basic conditions (7 M NH3/
of 2′-O-acetylthiomethyl-N6-phenoxyacetyl adenosine previ- MeOH; nBuNH2 in THF; NaOMe/MeOH; KOH/MeOH) have been
Scheme 2. Synthesis of S-(2′-O-methyladenosyl) 5′-thioadenosine 1. A = adenine. APac = N6-phenoxyacetyl adenine.
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screened to give the thiolate derivatives of 7 or 8, and to com- addition of three equivalents of oxidant in three portions. HPLC
pare the coupling efficiency with 6. It is worth mentioning that analysis of the crude material exhibited two peaks with a 68/
the 5′-thioacetyl adenosine 8 was totally converted into its 32 ratio corresponding to the sulfoxide 11 and the sulfone 12,
thiolate derivative within short reaction times from 10 min with respectively. Dinucleosides 11 and 12 were isolated after purifi-
KOH/MeOH to 30 min with other basic conditions whereas the cation with 30 % and 21 % yield, respectively. Then, 11 and 12
5′-thiolate of 2′,3′-protected adenosine 7 was only formed at were deprotected with a fluoride treatment to give the sulfox-
50 % within 2 h with NH3/MeOH or 16 h with NaOMe or KOH ide dinucleoside 2 with 38 % yield and the sulfone dinucleoside
in MeOH. Consequently, the coupling was more efficient with 3 with 32 % yield after purification by C18 reversed-phase chro-
5′-thiolate from 8 than from 7. The 5′,3′-TIPDS dinucleoside 10 matography. HPLC analysis of 2 exhibits two peaks with 74:26
was obtained with 61 % yield from the coupling of potassium ratio corresponding to the two (R) and (S) diastereoisomers. No
thiolate salt of 8 with the chloromethyl nucleoside 6 in a mix- attempt was made to determine the absolute stereochemistry
ture dichloromethane/methanol after 2 h reaction at room tem- at the sulfur atom. The sulfoxide-linked dinucleoside 2 will be
perature. The last step was the removal of the TIPDS group with first evaluated as a diastereoisomeric mixture in the inhibition
a fluoride ions treatment for 2 h at 50 °C to release the S-(2′-O- assays.
methyladenosyl) 5′-thioadenosine 1 with 89 % yield and high
purity after purification by C18-reversed-phase silica gel chro-
matography.
Synthesis of adenine dinucleosides 2 and 3 with sulfox-
ide- or sulfone-containing linkers. To extend the series of
thioether-linked dinucleoside, sulfoxide- or sulfone-containing
linkers were evidently designed. The ease of preparation of
oxidized sulfides and the potential increase of affinity for en-
zymes by allowing two extra H-bonds between the oxygen of
Synthesis of adenine dinucleoside 4 with an ethylthio-
ether linker. The dinucleoside 4 with a longer ethylthioether
linker than the one of 1 was synthesized by coupling the 2′-O-
modified adenosine 13 with an electrophile site at 2′-position
and the 5′-thiolate derivative from 8. Thus, the 2′-O-(tosylethyl)
adenosine 13 was prepared in four steps from adenosine
following a described procedure for the three first steps
(Scheme 4).[23]
The first step has consisted in introducing a methyl ester
S=O and these two hydrogen bonds prompted us to oxidize group preferentially at 2′-position whereas the 3′-OH and 5′-OH
the sulfur atom of dinucleoside 1 into sulfoxide 2 or sulfone 3 were unprotected. The reaction was conducted in the presence
derivatives (Scheme 3). Selective oxidation of the sulfide can be of NaH and the 2′-O-(methoxycarbonylmethyl) adenosine was
performed with several oxidizing agents such as m-chloroper- the main compound isolated with satisfactory 42 % yield. The
benzoic acid (m-CPBA), sodium periodate, potassium hydrogen 3′- and 5′-isomers were also formed at a lower extent. The next
persulfate (oxone®). However, m-CPBA was not soluble in the step was the masking of 3′-OH and 5′-OH by tert-butyldimethyl-
THF/MeOH/H2O solution mixture required for dissolution of the silyl (TBS) groups with 83 % yield. The reduction of the ester
substrate 1 and sodium periodate led to the oxidizing cleavage function in the presence of LiAlH4 gave the 5′,3′-O-TBS 2′-O-
of the cis-diol-containing adenosine as a side reaction. Finally, (2-hydroxyethyl) adenosine quantitatively. Unlike reported work
oxone® was selected for its ease of use, stability, non-toxicity described the alcohol activation with a mesylate group,[23] in
and solubility in the reaction mixture. The reaction with 3′,5′- our case the mesylate derivative was unstable and the coupling
TIPDS dinucleoside 10 was complete after 3 h at 0 °C after between both adenosines did not succeed. In contrast, the tos-
Scheme 3. Synthesis of S-(2′-O-methyladenosyl) 5′-sulfoxide adenosine 2 and S-(2′-O-methyladenosyl) 5′-sulfone adenosine 3.
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Scheme 4. Synthesis of S-(2′-O-ethyladenosyl)-5′-thioadenosine 4.
ylate derivative 13 obtained with 86 % yield was stable and was pyridine adenosine A2 in basic conditions with release of 2-
treated with the 5′-potassium thiolate adenosine from 8 to give thio-5-nitropyridine. In a first attempt, the coupling was per-
the 2′,5′-ethylthioether-linked dinucleoside 14 (19 %).[24] This formed between one equivalent of 2′-O-methylthioacetyl
low yield would have been improved by addition of 18-crown- N6-Pac-adenosine 16[21] (at 58 m
M concentration) obtained
6 ether to increase the reactivity of the thiolate nevertheless from the chloromethyl derivative 6, and an excess (1.2 equiv.)
preliminary assays had shown that the separation by gel chro- of 5′-disulfanylnitropyridine adenosine 18 prepared from 5′-
acetylthioadenosine 8 (Scheme 5).[22a] The reaction was carried
out at 0 °C in the presence of 7 M ammonia in methanol and
after 15 min, three peaks were noticed in the reverse-phase
HPLC chromatogram with a 35:45:20 ratio (Figure S14). These
three major peaks have been assigned to three dinucleosides
matography of 14 from 18-C-6 ether could not be achieved.
Finally, an Et3N. 3HF treatment was applied to remove the TBS
groups from 14 to afford the dinucleoside 4 in 44 % yield after
purification.
Synthesis of adenine dinucleoside with a disulfide linker
5. To generate the disulfide bridge between two adenosines, a with the linker attached at different positions in the adenosines
thiol-disulfide exchange reaction has been intended between a (Scheme S1). The main peak corresponds to the desired dinu-
thiolate derivative at 2′-position of A1 and a 5′-disulfanylnitro- cleoside 5 with a 2′,5′-disulfide linker. The other peaks were
Scheme 5. Synthesis of S-(2′-O-methylthioadenosinyl)-5′-thioadenosine 5.
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Figure 2. Reverse-phase HPLC profile of the crude mixture after the coupling reaction between 17 (23 mM) and 18 (1.80 equiv.).
assigned to the symmetrical dinucleosides with 2′,2′- or 5′,5′- contaminant resulting from the total deprotection of 17 in basic
disulfide-linker resulting from the coupling of synthon 16 or medium during the coupling reaction.
synthon 8 with themselves, respectively.
Finally, dinucleoside 19 was engaged in the last deprotection
Next, the conditions of the coupling reaction have been opti- step to remove the DMTr group with 80 % acetic acid in water
mized to obtain the dinucleoside 5 with an improved yield. to give the disulfanyl-linked dinucleoside 5 that was isolated
Either a higher (91 m
16 were tested and it was shown that a diluted solution of 16 15 % yield over 2 steps (coupling and deprotection).
at 23 m was favorable to a high proportion (73 %) of 5 in the
M) or a lower (23 mM) concentration for with high purity after purification by C18-chromatography in
M
crude mixture. In contrast, increasing the amount of 18 up to
3 equivalents rather promoted the formation of the 5′,5′-disulf-
ide link dinucleoside. However, even though in the optimized
conditions, the dinucleoside 5 was the major compound in the
mixture, we were not able to isolate 5 with high purity and
in sufficient amount due to a delicate separation of the three
dinucleosides which may be explained by the similarity of their
structure. To improve this separation, we introduced the lipo-
philic dimethoxytrityl (DMTr) group at 5′-position of 16 to give
5′-O-DMTr 2′-O-SAc adenosine 17. This nucleoside 17 used at
Conclusion
In this paper, we report the synthesis of five adenine dinucleo-
sides with S-linkers as bisubstrate SAM analogues for 2′-O-
methyltransferases that catalyze the 2′-O-methylation of the 5′
cap of viral mRNA or at internal positions within RNA sequence.
These bisubstrates contain a 5′-thioadenosine mimicking the
SAM adenosine and attached to the 2′-position of another
adenosine through an alkyl S-linker, mimicking the 5′-end of
RNA substrate. Such analogues were designed as mimics of the
transition state of the 2′-O-methylation of RNA with both part-
ners of the reaction. The evaluation of the bisubstrate ana-
logues as inhibitors of various 2′-O-MTases of emerging viruses
(Zika, Dengue, Ebola, SARS, MERS) is currently in progress.
Moreover, the S-linked adenine dinucleosides are valuable tools
to start structural studies on viral MTases before further studies
with short RNAs incorporating the bisubstrate molecules at
5′-end or at internal positions. Rationally, the prospects of this
work stand the synthesis of new bisubstrates with other hetero-
atom-containing linkers therefore the synthesis of analogues
with amine-type linkages (OCH2CH2NR2) is ongoing.
23 m
M
concentration was treated with 1.8 equivalent of 18 in
7 M NH3/MeOH to give the three dinucleosides as previously.
However, the ratio was different and the benefit of the lipophil-
icity of DMTr group was crucial for the separation since the
dinucleoside with 2′,2′-disulfide linker is DMTr-protected at
both 5′-positions (Rt 15.66), the dinucleoside with 5′,5′-disulfide
linker is the most polar with both 5′-OH (Rt 4.29) and the di-
nucleoside 19 has only one DMTr group at 5′-position of A1 (Rt
10.68) (Figure 2). It is noteworthy that to get rid of the remain-
ing excess of 5′-nitropyridinyl disulfide adenosine 18 by re-
verse-phase chromatography the use of a 50 mM triethylammo-
nium acetate buffer pH7 instead of water as eluent was recom-
mended to avoid contamination of all the fractions by the nitro-
pyridine derivative. Indeed, the pyridine moiety exists under
protonated/deprotonated equilibrium in water and 18 spreads
out all over the column chromatography. After purification,
HPLC analysis showed that the isolated compound 19 was 75 %
Experimental section
General Methods: DIEA was distilled from calcium hydride. All dry
solvents and reagents were purchased from commercial suppliers
pure and 5′-O-DMTr adenosine was characterized as the main and were used without further purification. Thin-layer chromatogra-
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phy (TLC) analyses were carried out on silica plate 60 F254. Purifica-
1 as a white powder (62 mg, 110 μmol, 89 %) with 99 % purity
tions by column chromatography were performed using Biotage determined by HPLC analysis at 260 nm. 1H-NMR (600 MHz,
Isolera 1 system with Column Flash Pure from Büchi. NMR experi- [D6]DMSO) δ = 8.35 (s, 1H, A1H8); 8.29 (s, 1H, A2H8); 8.13 & 8.14 (2s,
ments were recorded on Bruker 400, 500 or 600 spectrometers at 2H, A1H2 & A2H2); 7.33 (s, 2H, A2NH2); 7.28 (s, 2H, A1NH2); 6.03 (d,
20 °C. HRMS analyses were obtained with electrospray ionization J = 5.9 Hz, 1H, A1H1′); 5.83 (d, J = 5.9 Hz, 1H, A2H1′); 5.47 (d, J =
(ESI) in positive mode on a Q-TOF Micromass spectrometer. Analyti- 6.1 Hz, 1H, A2OH2′); 5.45 (m, 1H, A1OH5′); 5.28 (d, J = 5.3 Hz, 1H,
cal HPLC was performed on a UHPLC Thermoscientific Ultimate
A1OH3′); 5.24 (d, J = 5.3 Hz, 1H, A2OH3′); 4.81 (d, J = 11.7 Hz, 1H,
3000 system equipped with a LPG-3400RS pump, a DAD 3000 de- OCH2S); 4.76 (t, J = 5.3 Hz, 1H, A1H2′); 4.69 (d, J = 11.7 Hz, 1H,
tector and an WPS-3000TBRS Autosampler, Column Oven TCC- OCH2S); 4.67 (q, J = 5.8 Hz, J = 11.2 Hz, 1H, A2H2′); 4.35 (m, 1H, A1H3′);
3000SD. Dinucleosides 1–5 were analyzed by RP-HPLC (Macherey
4.03 (m, 1H, A2H3′); 3.98 (q, J = 3.4 Hz, J = 6.9 Hz, 1H, A1H4′); 3.94
Nagel Nucleodur C18 3 μm, 4.6 × 75 mm). The following HPLC sol- (m, 1H, A2H4′); 3.69 & 3.56 (2m, 2H, A1H5′ & A1H5′′); 2.73 (dd, J =
vent systems were used: 1 % CH3CN in 12.5 m
M
TEAAc (buffer A),
1.4 Hz, J = 6.7 Hz, 2H, A2H5′& A2H5′′). 13C-NMR (150 MHz, [D6]DMSO)
δ = 156.14 & 156.05 (A1C6 & A2C6); 152.7 & 152.5 (A1C2 & A2C2);
149.4 (A2C4); 148.9 (A1C4); 139.7 (A2C8); 139.6 (A1C8); 119.3 (A2C5);
119.1 (A1C5); 87.3 (A2C1′); 86.3 (A1C4′); 86.1 (A1C1′); 83.6 (A2C4′); 78.2
(A1C2′); 72.7 (OCH2S); 72.6 (A2C2′); 72.5 (A2C3′); 68.9 (A1C3′); 61.4
(A1C5′); 32.2 (A2C5′). HRMS (ESI+): m/z calcd. for C21H27N10O7S [M +
H]+: 563.1785, found 563.1786.
80 % CH3CN in 12.5 m TEEAc (buffer B). Flow rate was 1 mL/min.
M
UV detection was performed at 260 nm. Lyophilized compounds 1–
5 were stored at –20 °C for several months without any degradation.
S-(3′,5′-(Tetraisopropyldisiloxane-1,3-diyl)-2′-O-methyladenos-
yl)-5′-thioadenosine (10): To a solution of 3′,5′-O-tetraisopropyl-
disiloxane 2′-O-methylthiomethyl N6-phenoxyacetyl adenosine[21]
(0.50 g, 0.71 mmol, 1.00 equiv.) in anhydrous CH2Cl2 (2.50 mL) was
S-(3′,5′-(Tetraisopropyldisiloxane-1,3-diyl)-2′-O-methyladenos-
yl)-5′-sulfoxide Adenosine (11) & S-(3′,5′-(Tetraisopropyldisilox-
ane-1,3-diyl)-2′-O-methyladenosyl)-5′-sulfone Adenosine (12):
Compound 10 (0.93 mg, 1.16 mmol, 1.00 equiv.) was suspended in
a mixture of THF (7 mL), MeOH (7 mL) and water (1.80 mL). After
sonication, NaHCO3 (0.378 g, 4.52 mmol, 3.90 equiv.) and oxone®
(0.177 g, 1.16 mmol, 1.00 equiv.) were added. After 1 h stirring at
room temperature under argon, an additional equivalent of oxone®
was added and the reaction mixture was stirred for an additional
hour. Another additional equivalent of oxone® was added and the
reaction mixture was stirred again for an additional hour. The solu-
tion was then quenched with saturated aqueous NaHCO3 (10 mL).
The aqueous layer was extracted with AcOEt (6 x 50 mL) and the
combined organic extracts were washed with saturated aqueous
NaCl (3 x 50 mL), dried with Na2SO4 and concentrated under vac-
uum. The residue was purified via chromatography (dry-loading).
Compound 12 was first eluted with 14 % MeOH in AcOEt and iso-
lated as a white solid (0.20 g, 0.239 mmol, 21 %). Compound 11
was isolated as a white solid (0.288 g, 0.351 mmol, 30 %) after elu-
tion with 18 % MeOH in AcOEt.
added 1.0
M sulfuryl chloride (SO2Cl2) in dichloromethane (1.10 mL,
1.06 mmol, 1.50 equiv.) diluted in anhydrous CH2Cl2 (2.50 mL) in a
dropping funnel. The reaction mixture was stirred for 2 h at room
temperature under argon. The solvent was removed under vacuum
and the crude mixture of 6 was diluted in anhydrous CH2Cl2 (3 mL).
In parallel, 5′-thioacetyl adenosine 8 (0.39 mg, 1.20 mmol,
1.70 equiv.) suspended in MeOH (2 mL) in a round flask was treated
with a solution of potassium hydroxide KOH (0.14 g, 2.49 mmol,
3.50 equiv.) in MeOH (4 mL). The reaction mixture was stirred for
30 min at 0 °C under argon. The chloromethyl derivative 6 was
directly added to the solution containing the potassium thiolate of
8 and the reaction mixture was stirred for 2 h at room temperature
under argon. The solvents were removed under vacuum, and the
resulting residue was purified by silica gel chromatography (dry-
loading) with a 0–15 % MeOH linear gradient in CH2Cl2 to give the
dinucleoside 10 as a white solid (0.35 g, 0.435 mmol, 61 %). Rf =
1
0.66 (MeOH/CH2Cl2, 15:85); H-NMR (400 MHz, [D6]DMSO) δ = 8.30
(s, 1H, A2H8); 8.16 (s, 1H, A1H8); 8.13 (s, 1H, A2H2); 8.06 (s, 1H, A1H2);
7.33 (s, 2H, A1NH2); 7.26 (s, 2H, A2NH2); 5.99 (s, 1H, A1H1′); 5.85 (d,
J = 5.7 Hz, 1H, A2H1′); 5.50 (d, J = 6.0 Hz, 1H, A2OH2′); 5.29 (d, J =
5.1 Hz, 1H, A2OH3′); 5.04 (m, 1H, A1H3′); 5.03 (d, J = 11.7 Hz, 1H,
OCH2S); 4.93 (d, J = 11.6 Hz, 1H, OCH2S); 4.81 (d, J = 5.1 Hz, 1H,
A1H2′); 4.77 (q, J = 5.7 Hz, J = 11.1 Hz, 1H, A2H2′); 4.14 (q, J = 4.9 Hz,
1
11: Rf = 0.30 (MeOH/CH2Cl2, 15:85). H-NMR (600 MHz, [D6]DMSO)
δ = 8.33; 8.31; 8.19; 8.18; 8.14; 8.13; 8.04; 8.04; 7.33; 7.28; 5.90; 5.59;
5.56; 5.45; 5.09; 5.06; 4.90; 4.82; 4.79–4.73; 4.39–4.31; 4.26–4.19;
4.05–3.96; 3.92; 3.90–3.82; 3.28–3.16; 1.11–0.80. 13C-NMR (150 MHz,
[D6]DMSO) δ = 156.11; 152.57; 152.46; 149.21; 149.17; 148.49;
148.47; 140.19; 139.85; 139.79; 119.34; 119.32; 119.28; 88.32; 88.01;
87.68; 87.45; 86.45; 85.88; 83.57; 80.35; 80.28; 79.18; 78.97; 78.75;
77.33; 76.62; 73.31; 73.23; 72.77; 72.74; 69.97; 69.91; 60.11; 51.22;
49.25; 17.29–11.99. HRMS (ESI+): m/z calcd. for C33H53N10O9SSi2:
821.32507, found 821.32287.
x
J = 9.0 Hz, 1H, A2H3′); 4.02–4.07 (m, 2H, A2H4′, A1H5 ); 3.90–3.99 (m,
2H, A1H4′, A1H5 ); 2.87–3.06 (m, 2H, A2H5′, A2H5′′); 0.96–1.03 (m, 28H,
x
HTIPDS). 13C-NMR (150 MHz, [D6]DMSO) δ = 156.11 & 156.09 (A1C6 &
A2C6); 152.6 & 152.5 (A1C2 & A2C2); 149.4 (A2C4); 148.5 (A1C4); 139.9
(A2C8); 139.5 (A1C8); 119.3 (A1C5); 119.2 (A2C5); 87.7 & 87.5 (A1C1′&
A2C1′); 83.5 (A2C4′); 80.7 (A1C4′); 77.3 (A1C2′); 72.72, 72.68, 72.57
(OCH2S, A2C2′, A2C3′); 69.7 (A1C3′); 60.1 (A1C5′); 32.1 (A2C5′); 17.32–
12.05 (CTIPDS). HRMS (ESI+): m/z calcd. for C33H53N10O8SSi2 [M, H]+:
805.3307, found 805.3317.
1
12: Rf = 0.50 (MeOH/CH2Cl2, 15:85). H-NMR (600 MHz, [D6]DMSO)
δ = 8.35 (s, 1H, AxH8); 8.18 (s, 1H, AxH8); 8.18 (s, 1H, AxH2); 8.03 (s,
S-(2′-O-Methyladenosyl)-5′-thioadenosine (1): To a solution of 10
1H, AxH2); 7.35 (s, 2H, AxNH2); 7.29 (s, 2H, AxNH2); 6.02 (s, 1H, A1H1′);
(0.10 g, 0.12 mmol, 1.00 equiv.) in anhydrous THF (6 mL) was added 5.91 (d, J = 5.5 Hz, 1H, A2H1′); 5.64 (d, J = 5.8 Hz, 1H, A2OH2′); 5.55
1 M Et3N-3HF solution in THF (60 μL, 3.72 mmol, 3.00 equiv.). After
stirring for 2 h at 50 °C, the reaction mixture was treated with 2 M
(d, J = 5.0 Hz, 1H, A2OH3′); 5.06 (dd, J = 9.2, 4.8 Hz, 1H, A1H3′); 4.96
(d, J = 4.8 Hz, 1H, A1H2′); 4.90 (d, J = 12.3 Hz, 1H, OCH2SO2); 4.78
triethylammonium acetate buffer (pH 7). The solvents were re- (q, J = 5.3 Hz, 1H, A2H2′); 4.63 (d, J = 12.3 Hz, 1H, OCH2SO2); 4.31
moved under vacuum then water (10 mL) and CH2Cl2 (10 mL) were (dt, J = 9.2, 3.5 Hz, 1H, A2H4′); 4.28–4.17 (m, 1H, A2H3′); 4.04–3.91
added. The aqueous layer was extracted three times with CH2Cl2
(m, 2H, A1H5′, A2H5′); 3.82–3.76 (m, 1H, A2H5′′); 3.71 (dt, J = 9.1,
and once with Et2O and was evaporated under vacuum. The result- 2.7 Hz, 1H, A1H4′); 3.59–3.49 (m, 1H, A1H5′′); 1.06–0.79 (m, 28H,
ing residue was purified by chromatography on reversed-phase sil- HTIPDS). 13C-NMR (150 MHz, [D6]DMSO) δ = 156.15 & 156.13 (A1C6 &
ica gel column C18 (4 g, 40 μm) with a 0–25 % acetonitrile linear
gradient in TEAAc buffer 50 m , pH 7. The fractions containing the
pure compound were pooled, concentrated and lyophilized to give
A2C6); 152.59 & 152.51 (A1C2 & A2C2); 149.20 (A2C4); 148.51 (A1C4);
139.96 & 139.94 (A1C8 & A2C8); 119.33 & 119.22 (A1C5 & A2C5); 88.08
(A2C1′); 87.14 (A1C1′); 83.72 (OCH2SO2); 83.11 (A1C2′); 80.03 (A1C4′);
M
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78.28 (A2C4′); 73.12 (A2C3′); 72.32 (A2C2′); 70.02 (A1C3′); 59.74 (A2C5′); washed with saturated aqueous NaHCO3. The aqueous layer was
52.76 (A1C5′); 17.27–12.03 (CTIPDS). HRMS (ESI+): m/z calcd. for
C33H53N10O10SSi2: 837.31999, found 837.31950.
extracted with CH2Cl2 (3 × 40 mL) and the combined organic ex-
tracts were washed with saturated aqueous NaCl (60 mL), dried
with Na2SO4 and concentrated under vacuum. The residue was puri-
fied by chromatography with a linear gradient 0–10 % MeOH in
CH2Cl2 yielding to 13 as a white solid (1.40 g, 2.02 mmol, 86 %). Rf
0.71 (MeOH/CH2Cl2, 5:95). 1H-NMR (500 MHz, CDCl3) δ = 8.30 (s, 1H,
H2); 8.15 (s, 1H, H8); 7.75 (d, J = 8.3 Hz, 2H, Hortho Ts); 7.29 (d, J =
8.0 Hz, 2H, Hmeta Ts); 6.02 (d, J = 3.4 Hz, 1H, H1′); 5.74 (s, 2H, NH2);
4.50 (dd, J = 5.7, 4.7 Hz, 1H, H3′); 4.31 (dd, J = 4.7, 3.5 Hz, 1H, H2′);
4.20–4.10 (m, 1H, CH2-OTs); 4.04 (m, 1H, H4′); 3.98 (dd, J = 11.5,
3.4 Hz, 1H, H5′ or H5′′); 3.85–3.81 (m, 2H, 2′O-CH2); 3.75 (dd, J = 11.5,
2.7 Hz, 1H, H5′ or H5′′); 2.42 (s, 3H, CH3 Ts); 0.91 & 0.89 (2s, 18H,
Si-C(CH3)3); 0.09– 0.06 (4s, 12H, Si-CH3). 13C-NMR (125 MHz, CDCl3)
δ = 155.3 (C6); 152.9 (C2); 149.5 (C4); 144.8 (Cq-CH3 Ts); 139.4 (C8);
132.8 (Cq – SO2C Ts); 129.8 (Cméta Ts); 128.0 (Cortho Ts); 120.1 (C5); 87.0
(C1′); 84.6 (C4′); 82.6 (C2′); 69.8 (C3′); 68.7 (CH2 – OTs); 68.3 (2′O – CH2);
61.6 (C5′); 26.0 & 25.7 (CH3)3 TBS); 21.6 (CH3 Ts); 18.5 & 18.1
(C(CH3)3 TBS); –4.6&-4.9 & –5.4 (CH3 TBS). HRMS (ESI+): m/z calcd. for
S-(2′-O-Methyladenosyl)-5′-sulfoxide Adenosine (2): To a solu-
tion of 11 (0.10 g, 0.12 mmol, 1.00 equiv.) in anhydrous THF (6 mL)
was added Et3N-3HF (60 μL, 3.66 mmol, 3.00 equiv.). After 2 h stir-
ring at 25 °C, the reaction mixture was treated with 2 M triethyl-
ammonium acetate buffer (pH 7). The solvents were removed under
vacuum then water (10 mL) and CH2Cl2 (10 mL) were added. The
aqueous layer was extracted three times with CH2Cl2 and once with
Et2O and was evaporated under vacuum. The resulting residue was
purified by chromatography on a C18 reversed-phase silica gel col-
umn (4 g, 40 μm) with a 0–25 % acetonitrile linear gradient in
50 mM TEAAc buffer, pH 7. The fractions containing the pure com-
pound were pooled, concentrated and lyophilized to give 2 as a
white powder (27 mg, 47 μmol, 39 %) with 98 % purity determined
by HPLC analysis at 260 nm. 1H-NMR (600 MHz, [D6]DMSO) δ = 8.35;
8.33; 8.32; 8.14; 8.13; 7.34; 7.30; 6.10; 6.07; 5.90; 5.59; 5.57; 5.54; 5.47;
5.44; 5.39; 4.85; 4.82; 4.72; 4.68; 4.61; 4.40; 4.36; 4.25; 4.20–4.14;
3.99; 3.66; 3.58–3.50; 3.30; 3.26–3.21; 3.18; 3.09. 13C-NMR (150 MHz,
[D6]DMSO) δ = 156.61; 156.56; 153.13; 153.03; 153.00; 149.77;
149.67; 149.45; 140.57; 140.35; 140.18; 140.13; 119.74; 119.68; 88.55;
88.32; 86.53; 86.36; 86.29; 85.45; 83.78; 83.39; 78.47; 77.92; 73.61;
73.45; 73.24; 73.01; 69.53; 69.42; 61.73; 51.29; 50.44. HRMS (ESI+):
m/z calcd. for C21H27N10O8S: 579.17286, found 579.17224.
C
31H52N5O7SSi2 [M + H]+: 694.3126, found 694.3121.
3′,5′-Bis-O-(tert-butyldimethylsilyl)-S-(2′-O-ethyladenosyl)-5′-
thioadenosine (14): To a suspension of 8 (135 mg, 0.42 mmol,
1.00 equiv.) in MeOH (4 mL) was added a solution of KOH (47 mg,
0.83 mmol, 2.00 equiv.) in MeOH (2 mL). After stirring for 20 minutes
at 0 °C, a solution of 13 (346 mg, 0.50 mmol, 1.20 equiv.) in CH2Cl2
(4 mL) was added and the reaction mixture was stirred for addi-
tional 2 h at room temperature. The solvents were removed under
vacuum, and the resulting paste was purified by chromatography
(dry-load) with a 0–15 % linear gradient MeOH in CH2Cl2 yielding
to 14 as a white solid (63 mg, 0.078 mmol, 19 %). Rf 0.67 (MeOH/
CH2Cl2, 15:85). 1H-NMR (400 MHz, [D6]DMSO) δ = 8.31 (s, 2H, A1H8 &
A2H8 ); 8.14 (s, 1H, AxH2); 8.13 (s, 1H, AxH2); 7.30 (s, 2H, AxNH2); 7.26
(s, 2H, AxNH2); 6.01 (d, J = 4.4 Hz, 1H, A1H1′); 5.86 (d, J = 5.7 Hz, 1H,
A2H1′); 5.48 (d, J = 6.0 Hz, 1H, A2OH2′); 5.27 (d, J = 5.1 Hz, 1H, A2OH3′);
4.70 (q, J = 5.7 Hz, 1H, A2H2′); 4.60 (m, 1H, A1H2′); 4.56 (m, 1H, A1H3′);
S-(2′-O-Methyladenosyl)-5′-sulfone Adenosine (3): To a solution
of 12 (0.10 g, 0.12 mmol, 1.00 equiv.) in anhydrous THF (6 mL) was
added Et3N-3HF (60 μL, 3.59 mmol, 3.00 equiv.). After 2 h stirring at
25 °C, the reaction mixture was treated with 2 M triethylammonium
acetate buffer (pH 7). The solvents were removed under vacuum
then water (10 mL) and CH2Cl2 (10 mL) were added. The aqueous
layer was extracted three times with CH2Cl2 and once with Et2O
and was evaporated under vacuum. The resulting residue was puri-
fied by chromatography on a C18 reversed-phase silica gel column
(4 g, 40 μm) with a 0–25 % acetonitrile linear gradient in 50 m
M
4.11–4.07 (m, 1H, A2H3′); 3.97–3.86 (m, 3H, A1H4′, A2H4′
2′OCH); 3.70–3.61 (m, 3H, 2′OCH, A1H5′H5′′); 2.91–2.77 (m, 2H, A2H5′
5′′); 2.63 (m, 2H, CH2S); 0.87 (s, 9H, SiC(CH3)3); 0.83 (s, 9H, SiC(CH3)3);
,
TEAAc buffer, pH 7. The fractions containing the pure compound
were pooled, concentrated and lyophilized to give 3 as a white
powder (23 mg, 39 μmol, 32 %) with 98 % purity determined by
HPLC analysis at 260 nm. 1H-NMR (600 MHz, [D6]DMSO) δ = 8.35 (s,
1H, A2H8); 8.31 (s, 1H, A1H8); 8.16 (s, 1H, A2H2); 8.14 (s, 1H, A1H2);
7.42–7.23 (m, 4H, A1NH2, A2NH2); 6.08 (d, J = 4.9 Hz, 1H, A1H1′); 5.91
(d, J = 5.3 Hz, 1H, A2H1′); 5.62 (d, J = 5.8 Hz, 1H, A2OH2′); 5.49 (d,
J = 5.2 Hz, 1H, A2OH3′); 5.38 (d, J = 5.3 Hz, 1H, A1OH3′); 5.35 (dd, J =
6.5, 4.7 Hz, 1H, A1OH5′); 4.90–4.83 (m, 1H, A1H2′); 4.75 (d, J = 12.6 Hz,
1H, OCH2SO2); 4.71–4.63 (m, 2H, OCH2SO2, A2H2′); 4.46–4.40 (m, 1H,
A1H3′); 4.28–4.23 (m, 1H, A2H4′); 4.21–4.16 (m, 1H, A2H3′); 3.95–3.89
(m, 1H, A1H4′); 3.73–3.68 (m, 1H, A2H5′); 3.68–3.65 (m, 1H, A1H5′);
3.56–3.49 (m, 1H, A1H5′′); 3.46–3.39 (m, 1H, A2H5′′). 13C-NMR
(150 MHz, [D6]DMSO) δ = 156.12 & 156.11 (A1C6 & A2C6); 156.69
(A2C2); 152.51 (A1C2); 149.23 (A2C4); 148.96 (A1C4); 139.86 (A2C8);
139.52 (A1C8); 119.24 (A1C5 & A2C5); 88.00 (A2C1′); 86.27 (A1C1′);
85.51 (A1C4′); 83.17 & 83.10 (A1C2′ & OCH2SO2); 77.76 (A2C4′); 72.93
(A2C3′); 72.41 (A2C2′); 68.88 (A1C3′); 61.06 (A1C5′); 52.47 (A2C5′). HRMS
(ESI+): m/z calcd. for C21H27N10O9S: 595.1678, found 595.1675.
H
0.09 (s, 3H, SiCH3); 0.07 (s, 3H, SiCH3); 0.02 (s, 3H, SiCH3); –0.00 (s,
3H, SiCH3). 13C-NMR (100 MHz, [D6]DMSO) δ = 156.0 (AxC6); 152.6
(AxC2); 149.4 & 149.1 (AxC4); 139.7 & 139.3 (AxC8); 119.1 (AxC5); 87.4
(A2C1′); 85.7 (A1C1′); 84.3 (A1C4′); 83.8 (A2C4′); 80.3 (A1C2′); 72.6 (A2C2′);
72.5 (A2C3′); 69.9 (A1C3′); 69.7 (A1C5′); 61.8 (2′OCH2); 34.3 (A2C5′); 31.3
(CH2S); –25.7& 25.6 (C(CH3)3 TBS); 17.9 & 17.76 (C(CH3)3 TBS); –4.8 &
–5.1 & –5.6 (CH3 TBS). HRMS (ESI+): m/z calcd. for C34H57N10O7SSi2
[M + H]+: 805.3671, found 805.3669.
S-(2′-O-Ethyladenosyl)-5′-thioadenosine (4): To a solution of 14
(63 mg, 0.078 mmol, 1.00 equiv.) in anhydrous THF (3.7 mL) was
added 1 M Et3N-3HF solution in THF (50 μL, 0.31 mmol, 4.00 equiv.).
After stirring for 3 h at 50 °C, the reaction mixture was treated with
2 M triethylammonium acetate buffer (pH 7). The solvents were
removed under vacuum before water (10 mL) and CH2Cl2 (10 mL)
were added. The aqueous layer was extracted three times with
CH2Cl2 and once with Et2O. The solvent was removed under vac-
uum and the resulting paste was purified by chromatography on a
reversed-phase C18 silica gel column (4 g, 40 μm) with a 0–25 %
3′,5′-Bis-O-(tert-butyldimethylsilyl)-2′-O-(2-toluene-sulfonyl-
ethyl) Adenosine (13): To a solution of 3′,5′-bis-O-(tert-butyldi-
linear gradient of acetonitrile in 50 mM TEAAc buffer, pH 7 followed
methylsilyl)-2′-O-(hydroxyethyl) adenosine[23] (1.23 g, 2.34 mmol, by concentration and lyophilization of pure fractions to give 4 as a
1.00 equiv.) in anhydrous CH2Cl2 (7 mL) was added successively 4-
dimethylaminopyridine (29 mg, 0.23 mmol, 0.10 equiv.), Et3N mined by HPLC analysis at 260 nm. H-NMR (600 MHz, [D6]DMSO)
white powder (20 mg, 0.035 mmol, 44 %) with 99 % purity deter-
1
(0.70 mL, 4.92 mmol, 2.10 equiv.) and 4-toluenesulfonyl chloride
(0.893 g, 4.68 mmol, 2.00 equiv.). After stirring for 4 hours at 0 °C
under argon, the solution was diluted with CH2Cl2 (30 mL) and
δ = 8.37 (s, 1H, A1H8); 8.33 (s, 1H, A2H8); 8.14 (2s, 2H, A1H2 & A2H2);
7.34 & 7.28 (2 br s, 4H, A1NH2 & A2NH2); 6.00 (d, J = 5.9 Hz, 1H,
A1H1′); 5.87 (d, J = 5.7 Hz, 1H, A2H1′); 5.49 (d, J = 6.1 Hz, 1H, A2OH2′);
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5.38 (dd, J = 6.9, 4.8 Hz, 1H, A1OH5′); 5.29 (d, J = 5.1 Hz, 1H, A2OH3′);
δ = 157.5 (A1C6); 157.2 (A2C6); 153.7 (A1C2); 153.7 (A1C2); 153.4
5.17 (d, J = 5.2 Hz, 1H, A1OH3′); 4.71 (q, J = 5.6 Hz, 1H, A2H2′); 4.50 (A2C2); 150.3 (A1C4); 149.6 (A2C4); 141.4 (A1C8); 140.7 (A2C8); 121.7
(m, 1H, A1H2′); 4.32 (m, 1H, A1H3′); 4.10 (m, 1H, A2H3′); 3.98 (m, 2H, (A1C5); 121.1 (A2C5); 90.4 (A2C1′); 89.2 (A1C1′); 88.6 (A1C4′); 83.5 (A2C4′);
A1H4′ & A2H4′); 3.72–3.65, 3.55–3.52 (m, 4H, A1H5′, A1H5′′, 2′OCH2); 81.2 (A1C2′); 81.1 (OCH2S); 74.5 (A2C2′); 73.9 (A2C3′); 71.2 (A1C3′); 63.3
2.90–2.79 (m, 2H, A2H5′, A2H5′′); 2.65 (m, 2H, CH2S). 13C-NMR (A1C5′); 42.8 (A2C5′). HRMS (ESI+): m/z calcd. for C21H27N10O7S2 [M +
(150 MHz, [D6]DMSO) δ = 156.14 &156.05 (A1C6 & A2C6); 152.65 &
152.52 (A1C2 & A2C2); 149.5 (A2C4); 149.0 (A1C4); 139.74 (A2C8);
139.66 (A1C8); 119.3 (A1C5); 119.1 (A2C5); 87.2 (A2C1′); 86.1 (A1C4′);
86.0 (A1C1′); 83.8 (A2C4′); 81.0 (A1C2′); 72.6 (A2C2′); 72.5 (A2C3′); 69.5
(A1C5′ or 2′OCH2); 68.9 (A1C3′); 61.3 (A1C5′ or 2′OCH2); 34.2 (A2C5′);
31.2 (CH2S). HRMS (ESI+): m/z calcd. for C22H29N10O7S [M + H]+:
577.1933, found 577.1933.
H]+: 595.1506, found 595.1505.
Acknowledgments
Rostom Ahmed-Belkacem thanks the University of Montpellier
for financial support.
S-(5′-O-(4,4′-Dimethoxytrityl-2′-O-methylthioadenosyl)-5′-thio-
adenosine (19): To a solution of 17[21] (202 mg, 0.26 mmol,
1.00 equiv.) in MeOH (3.5 mL) was added 18[22a] (200 mg,
0.46 mmol, 1.80 equiv.) and an ammonia solution (7 M in MeOH)
(7.5 mL). After stirring for 20 min at –10 °C under argon, the solvents
were removed and the resulting residue was purified by chromatog-
raphy (dry-load) on reversed-phase silica gel column C18 with a 20–
Keywords: Bisubstrate · RNA 2′-O-methylation · Transition
states · Viral RNA methyltransferases · Nucleosides
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Ryu), Academic Press, Boston, 2017, pp. 289–302.
[2] a) D. W. Leung, G. K. Amarasinghe, Curr. Opin. Struct. Biol. 2016, 36, 133–
141; b) E. Decroly, B. Canard, Curr. Opin. Virol. 2017, 24, 87–96; c) M.
Ringeard, V. Marchand, E. Decroly, Y. Motorin, Y. Bennasser, Nature 2019,
565, 500–504.
70 % linear gradient of acetonitrile in 50 mM TEAAc buffer, pH 7.
The fractions containing 19 with more than 75 % purity were
pooled and concentrated to dryness. Traces of TEAAc salts were
removed by several co-evaporations with water and acetonitrile to
give compound 19 (92 mg, 0.102 mmol, 40 % corrected yield) with
75 % purity determined by HPLC analysis at 260 nm. The main con-
taminant was the 5′-O-DMTr adenosine. Full characterization of 19
was performed with a 99 % pure fraction isolated after purification.
1H-NMR (400 MHz, 1,4-[D8]Dioxane) δ = 8.16 (s, 1H, H2 or H8); 8.14
(s, 1H, H2 or H8); 8.01 (s, 1H, H2 or H8); 7.95 (s, 1H, H2 or H8); 7.43–
7.13 (m, 9H, HDMTR); 6.78 (m, 4H, HDMTR); 6.47 (br s, 4H, NH2); 6.15
(d, J = 3.7 Hz, 1H, A1H1′); 5.84 (d, J = 4.4 Hz, 1H, A2H1′); 5.05 (m, 2H,
O-CH2-S); 4.93 (m, 1H, A1H2′); 4.82–4.66 (m, 2H, A2OH3′, A2H2′); 4.49
(d, 1H, A1H3′); 4.42–4.34 (m, 1H, A2OH2′); 4.29 (m, 1H, A2H3′); 4.23–
4.16 (m, 1H, A2H4′); 4.16–4.11 (m, 1H, A1H4′); 4.03 (d, J = 7.2 Hz, 1H,
A1OH3′); 3.73 (s, 6H, OCH3DMTR); 3.42–3.37 (m, 2H, A1H5′H5′′); 3.08 (d,
J = 6.3 Hz, 2H, A2H5′H5′′). 13C-NMR (150 MHz, 1,4-[D8]Dioxane) δ =
159.5 (Cq-OCH3 DMTR); 157.0 et 157.1 (A1C5 et A2C5); 153.7 et 153.9
(A1C6 et A2C6); 150.3 (A1C4 et A2C4); 146.1 (Cq DMTR); 140.2 et 140.6
(A1C8 et A2C8); 136.8 (Cq DMTR); 127.5, 128.5, 129.0, 130.9 (CH DMTR);
120.8 et 120.9 (A1C2 et A2C2); 113.9 (CHDMTR); 90.3 (A1C1′); 87.7 (A2C1′);
87.1 (O-Cq DMTR); 84.4 (A1C4′); 83.6 (A2C4′); 81.2 (O-CH2-S); 80.5 (A1C2′);
74.5 (A2C2′); 73.8 (A2C3′); 70.7 (A1C3′); 64.2 (A1C5′); 55.3 (O-CH3); 42.8
(A2C5′). HRMS (ESI–): m/z calcd. for C42H43N10O9S2 [M – H]–:
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S-(2′-O-Methylthioadenosyl)-5′-thioadenosine (5): The mixture
of 19 and the by-product 5′-O-DMTr adenosine was treated with a
solution of 80 % acetic acid in water (8.76 mL) and stirred for 15 min
at room temperature. The mixture solution was washed with CHCl3
(10 × 5 mL) then Et2O (1 × 10 mL). The solvent was removed under
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8.19 (s, 1H, A1H2); 8.18 (s, 1H, A2H2); 7.98 (s, 1H, A1H8); 7.97 (s, 1H,
A2H8); 6.62 (br s, 2H, NH2); 6.50 (br s, 2H, NH2); 6.00–5.95 (m, 2H,
A1H1′, A1OH5′); 5.86 (d, J = 4.3 Hz, A2H1′); 4.99 (m, 1H, A1H2′); 4.82
(s, 2H, OCH2S); 4.80 (m, 2H, A2H2′, A2OH2′); 4.44 (m, 2H, A2OH3′,
A2H3′); 4.30 (m, 1H, A2H3′); 4.12 (m, 2H, A1H4′, A2H4′); 3.99 (d, J =
4.2 Hz, A1OH3′); 3.84–3.59 (m, 2H, A1H5′, A1H5′′); 3.00 (dd, J = 1.7 Hz,
J = 5.9 Hz, 2H, A2H5′, A2H5′′). 13C-NMR (125 MHz, 1,4-[D8]Dioxane)
Eur. J. Org. Chem. 2019, 6486–6495
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© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

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Received: July 31, 2019
Eur. J. Org. Chem. 2019, 6486–6495
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© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim