Chemical Synthesis of Oligoribonucleotide (ASL of tRNALys T. brucei) Containing a Recently Discovered Cyclic Form of 2-Methylthio-N6-threonylcarbamoyladenosine (ms2ct6A)

Article abstract of DOI:10.1002/chem.201902411

The synthesis of the protected form of 2-methylthio-N⁶-threonylcarbamoyl adenosine (ms²t⁶A) was developed starting from adenosine or guanosine by using the optimized carbamate method and, for the first time, an isocyanate route. The hypermodified nucleoside was subsequently transformed into the protected ms²t⁶A-phosphoramidite monomer and used in a large-scale synthesis of the precursor 17nt ms²t⁶A-oligonucleotide (the anticodon stem and loop fragment of tRNA^Lys from T. brucei). Finally, stereochemically secure ms²t⁶A→ms²ct⁶A cyclization at the oligonucleotide level efficiently afforded a tRNA fragment bearing the ms²ct⁶A unit. The applied post-synthetic approach provides two sequentially homologous ms²t⁶A- and ms²ct⁶A-oligonucleotides that are suitable for further comparative structure–activity relationship studies.

Full text of DOI:10.1002/chem.201902411

A Journal of
Accepted Article
Title: Chemical synthesis of oligoribonucleotide (ASL of tRNALys
T. brucei) containing a recently discovered cyclic form of 2-
methylthio-N6-threonylcarbamoyladenosine (ms2ct6A)
Authors: Katarzyna Debiec, Michal Matuszewski, Karolina Podskoczyj,
Grazyna Leszczynska, and Elzbieta Sochacka
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To be cited as: Chem. Eur. J. 10.1002/chem.201902411
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10.1002/chem.201902411
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Chemical synthesis of oligoribonucleotide (ASL of tRNALys
T. brucei) containing a recently discovered cyclic form of
2-methylthio-N6-threonylcarbamoyladenosine (ms2ct6A)
Katarzyna Debiec,[a] Michal Matuszewski,[a] Karolina Podskoczyj,[a] Grazyna Leszczynska, [a] and
Elzbieta Sochacka *[a]
Abstract:
The synthesis of the protected form of 2-methylthio-N6-
threonylcarbamoyl adenosine (ms2t6A) was developed starting from
adenosine or guanosine using the optimized carbamate method and,
for the first time, the isocyanate route. The hypermodified nucleoside
was subsequently transformed to the protected ms2t6A–
phosphoramidite monomer and used in a large scale synthesis of
the precursor 17nt ms2t6A-oligonucleotide (the anticodon stem and
loop fragment of tRNALys from T. brucei). Finally, stereochemically
secure ms2t6A→ms2ct6A cyclization at the oligonucleotide level
afforded efficiently tRNA fragment bearing the ms2ct6A unit. Applied
post-synthetic approach provides two sequentially homologous
ms2t6A- and ms2ct6A-oligonucleotides suitable for further
comparative structure-activity relationship studies.
Figure 1. Structures of L-threonylcarbamoyl modified adenosines located at
position 37 of tRNAs
Recently, due to a remarkable development of biochemical
Introduction
and analytical methods, other analogs of t6A have been
identified in the tRNA anticodon loops, i.e. a t6A “linear”
Transfer RNAs (tRNAs) constitute a unique subset of
natural RNA, because in all domains of life these relatively short
derivative having a hydroxylated threonine methyl group19 (ht6A,
Figure 1) and two “cyclic” species bearing a hydantoin ring - N6-
biopolymers utilize, in total, more than 130 structurally diverse
threonylcarbamoyl-adenosine (ct6A)20,21 and 2-methylthio-N6-
analogs of canonical nucleosides.1-3 Their impact on the
threonylcarbamoyl adenosine (ms2ct6A).22 It was documented
biological processes (mostly related to the decoding of the
that ct6A and ms2ct6A are formed intracellularly upon enzymatic
genetic code) is a matter of intense studies.4-10 Interestingly, the
dehydration of “linear” t6A and ms2t6A units.20,22 Importantly, the
modifications are not distributed evenly along the tRNA
hydantoin derivatives were identified in several tRNAs in which
molecules11 and predominantly occupy positions 34 (the wobble
the "linear" t6A and ms2t6A were previously detected.20-22 This
position) and 37 (3′ side downstream from the anticodon) in the
initial misidentification resulted from high susceptibility of ct6A
anticodon loop and stem domains.1,5-7,11,12
and ms2ct6A to hydrolysis,20-23 (leading to t6A and ms2t6A units,
Among the modified nucleosides found at the position 37,
special interest is paid to N6-threonylcarbamoyladenosine (t6A37,
Figure 1) and its derivatives (ms2t6A and m6t6A, vide infra),
present in almost all known tRNAs decoding the A-starting
codons (ANN).1,11 The most abundant, namely t6A37 has been
intensively studied (for already 50 years13,14) because of diverse
functions it plays during protein biosynthesis.15 In some tRNAs,
t6A37 is replaced with an analog containing either an -SMe group
attached to the purine C2 atom (ms2t6A)16,17 or a methyl
substituent at the N6-atom (m6t6A).18
respectively) under alkaline conditions commonly used in the
standard protocol of tRNA isolation.24 Thus, the reported
existence of “linear” t6A/ms2t6A in tRNA molecules must be
verified and the results may lead to new ideas regarding the
biological functions of tRNAs bearing “cyclic” analogs.
Structural studies revealed that the N6-threonylcarbamoyl
chain in t6A adopts a planar alignment in respect to the adenine
ring.25.26 The same phenomenon was observed in the t6A-
containing RNA oligomers27-31 and that planarity is claimed to be
a crucial factor for proper strength of the codon-anticodon
interactions27-32 However, X-ray studies of the ct6A nucleoside
revealed a twisted arrangement of the hydantoin ring and the
adenine moiety, most likely enforced by strong electrostatic
repulsion between the carbonyl oxygen atoms of hydantoin and
the N1 and N7 nitrogen atoms of adenine.20 It is highly possible
that the hydantoin and adenine rings are also not coplanar in
biological milieu, with still unknown consequences for the
translation process. Thus, to investigate structural aspects and
biological functions of the tRNA containing ct6A or ms2ct6A
units, a reliable chemical method for the synthesis of suitable
model oligonucleotides is highly desired.
[a]
K. Debiec, Dr. M. Matuszewski, K. Podskoczyj, Dr. G. Leszczynska,
Prof. E. Sochacka,
Institute of Organic Chemistry, Faculty of Chemistry
Lodz University of Technology
Zeromskiego 116, 90-924 Łódź, Poland
e-mail: elzbieta.sochacka@p.lodz.pl
http://trna-group.p.lodz.pl/
Supporting information for this article is given via a link at the end of
the document.
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10.1002/chem.201902411
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Recently, we have reported a successful transformation
of t6A into ct6A and a method suitable for that cyclization at the
oligonucleotide level.33 Here we present the first introduction of
an ms2ct6A unit into a 17-mer with the sequence of anticodon
stem and loop (ASL) fragment of tRNALys from T. brucei, where
ms2ct6A was found as a native modification.22
material, and successfully combined synthetic methods already
reported in several sources.44-49 In brief, adenosine was
converted into adenosine N-oxide,44 which was subjected to the
opening of the 6-membered ring (5 M NaOH) with the formation
of 5-amino-1-(β-D-ribofuranosyl)imidazole-4-carboxamidoxime,
and later to the ring closure/thiolation (a reaction with CS2),
followed by simple S-methylation with MeI.44-46 Final acetylation
of the ribose hydroxyl groups yielded 1 in 31 % overall yield (for
experimental details see Scheme S1, Supporting Information).
The same protected nucleoside 1 was also obtained in a four-
step synthesis from guanosine (in 36 % overall yield), involving
acetylation of the ribose hydroxyl groups of nucleoside, followed
by a C6=O→C6-Cl conversion with POCl3,47 incorporation of the -
SMe group at the C2 atom and the final C6-Cl→C6-NH2
conversion48,49 (Scheme S2, Supporting Information).
Results and Discussion
Although chemical synthesis of RNA oligomers
containing the t6A or ms2t6A units by the phosphoramidite
approach34-36 using the ultra-mild protected monomers is
feasible,37-43 this approach is inappropriate for synthesis of RNAs
bearing ms2ct6A due to the aforementioned instability of the
hydantoin ring in even slightly alkaline media. Therefore, we
decided to explore an alternative post-synthetic methodology in
which the ms2t6A→ms2ct6A cyclization is performed in the
already assembled oligoribonucleotide containing the ms2t6A
unit. First, we optimized the synthesis of the 5’-O-DMTr-2′-O-
TBDMS protected “linear” ms2t6A nucleoside (protected at the
threonine –OH and –COOH functions) and made its 3’-O-
phosphoramidite derivative (5, Scheme 1). The monomer was
then used in the synthesis of an ms2t6A-modified oligonucleotide
(Figure 2). Finally, we performed an effective and
stereochemically safe conversion of ms2t6A→ms2ct6A at the
oligonucleotide level (Figure 2 and 3).
To modify the nucleobase in Ac3ms2A with
a
threonylcarbamoyl residue at the N6 position we applied a
carbamate or an isocyanate route (Scheme 1, path I and II,
respectively). The former is advantageous when the attachment
of an unprotected L-threonine residue (in a ureido fashion) is the
synthetic goal,50-52 but in the synthesis of 5, the phosphitylation
reaction must be preceded by an appropriate protection of the
hydroxyl and carboxylic groups. In the reported methods, the
tert-butyldimethylsilyl group (TBDMS) was used to protect the
hydroxyl function, and the carboxylic group was converted into a
base-labile 2-(4-nitrophenyl)ethyl ester (pNPE)38,41 or a fluoride-
sensitive trimethylsilylethyl ester (TMSE).37,39,40,42 Within the
TBDMS/pNPE protection scheme, O-tert-butyldimethylsilyl-L-
threonine 2-(4-nitrophenyl) ethyl ester was reacted with ms2A
carbamate,38 while the TBDMS/TMSE protection was carried out
in the already threonylated ms2t6A nucleoside by the reaction
with TBDMS triflate, followed by esterification with
2-(trimethylsilyl)ethanol in the presence of DCC.40,42
Optimization of ms2t6A phosphoramidite synthesis
In our preparation of the ms2t6A monomer 5, 2′,3′,5′-tri-O-
acetyl derivative of ms2A (Ac3ms2A, 1, Scheme 1) was a key
intermediate. This nucleoside was previously synthesized by the
D. R. Davis group with the use of the silyl method of N-glycosidic
bond formation between 2-methylthioadenine (not commercially
available, obtained with 4 steps synthesis from thiourea and
malonitrile) and 1-O-acetyl-2,3,5-tri-O-benzoyl-β-D-ribofuranose,
followed by the conversion into 2′,3′,5′-tri-O-acetyl derivative.41 In
our approach we used adenosine or guanosine as the starting
Scheme 1. Synthesis of ms2t6A phosphoramidite i) phenoxycarbonyl tetrazole (3.0 equiv), 1,4-dioxane, 40 oC, 20 h, 78 % yield; ii) pyridine, 40 oC, 24 h, 75 %
yield; iii) toluene, reflux, 16 h, 83 % yield; iv) triethylamine in methanol (10 % solution), rt, 24 h, 86 % yield; (synthetic steps from G to 1 and from A to 1 as well as
the procedures for the nucleoside 4 protection with DMTr and TBDMS groups and phosphitylation are described in details in Supporting Information).
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Synthesis of an ms2t6A-containing RNA oligonucleotide by
phosphoramidite chemistry
Manual synthesis of a 17nt oligoribonucleotide of the
sequence 5’-CACGGCUUUUms2t6AACCGUG-3’ (ms2t6A-ASL,
Figure 2) was performed on a CPG-support at a 2.5 µmol scale
using the modified Sproat’s protocol.35 The phosphoramidite
monomers other than 5, as well as guanosine attached to the
solid support, were N-protected with a 4-tert-butylphenoxyacetyl
(tac) groups to make the final deprotection safe for the relatively
base labile carbamoyl linkage in ms2t6A nucleoside. Also, the
oxidation step was modified to protect the 2-methylthio group in
the ms2t6A unit. Whereas in the previous synthesis of an
ms2t6A-oligonucleotide 10 % tBuOOH in acetonitrile was applied
as the oxidizing agent,41,42 we used a diluted 0.02 M iodine
solution in THF/H2O/pyridine, which we effectively and safely
used in synthesis of oligonucleotides containing the highly
oxidation sensitive 2-thiouridines.54-56 In the coupling steps
Scheme 2. Synthesis of L-threonine component i) tert-butyldimethylsilyl
chloride (2.2 equiv), imidazole (2.2 equiv), pyridine, 24 h, 98
ii) 2-(trimethylsilyl)ethanol (1.1 equiv), DCC (1.6 equiv), DMAP (0.1 equiv)
dichloromethane, h, 98 yield; iii) trifluoroacetic acid in
oC,
% yield;
0
3
%
dichloromethane (60 % solution), dichloromethane, rt, 5 min, 64 % yield; iv)
1,4-dioxane saturated with hydrogen chloride (4 M), dichloromethane, 5 min,
100 % yield; v) phosgene in toluene (20 % solution), pyridine, dichloromethane,
0 °C, 2 h, (100 % conversion observed for crude 7 from 6a)
(lasting for
phosphoramidites
8
min), 0.1
in dry
M
solutions of canonical
We explored the carbamate route (Scheme 1, path I)
using synthesized for the first time TBDMS/TMSE protected
L-threonine (6), which was obtained starting from commercially
available N-Boc-L-threonine (8, Scheme 2 and Experimental
Section). Compound 8 was almost quantitatively converted into
acetonitrile and 5-(3,5-
bis(trifluoromethyl)phenyl)-1H-tetrazole (0.25 M solution in ACN)
as an activator were used. To increase the efficiency of
incorporation of the ms2t6A unit, double coupling of 5 was
performed, each lasting for 25 min. Next, the protected ms2t6A-
ASL (still CPG-bound) was treated with TEA–ACN (1:1, v/v) to
remove the 2-cyanoethyl groups, followed by 8 M ethanolic
ammonia to remove the N-protecting groups and to release the
oligoribonucleotide from the support. The resultant solution was
concentrated in vacuo and treated with 1 M TBAF in THF to
remove the TBDMS and TMSE groups. The deprotected RNA
was isolated by IE-HPLC (for details see Experimental Section
and Supporting Information, Figure S2). The eluate was
concentrated to a small volume, desalted and lyophilized to yield
94 OD of ms2t6A-ASL, the structure of which was confirmed by
MALDI-TOF MS (Figure S3).
an
O-TBDMS
derivative
9,
then
esterified
with
2-(trimethylsilyl)ethanol in the presence of DCC, and, finally,
N-deprotected with trifluoroacetic acid to furnish the
corresponding salt 6 in 61 % overall yield. It was further coupled
with an active carbamoyl derivative 2 (Scheme 1), which was
prepared
in
the
reaction
of
2′,3′,5′-tri-O-acetyl-2-
methylthioadenosine (1) with phenoxycarbonyl tetrazole
according to the reported procedure.41,42 The condensation gave
fully protected ms2t6A (3) in 75 % yield. The same intermediate
3 was obtained on the isocyanate path II (Scheme 1). To this
goal, compound 6 (or 6a, Scheme 2) was treated with a
commercially available solution of phosgene in toluene at 0 °C,
according to the racemization-free Nowick’s procedure.53 After
short aqueous workup (to avoid hydrolysis) an isocyanate
derivative 7 was obtained and reacted (without purification) with
2′,3′,5′-tri-O-acetyl-2-methylthioadenosine (1) to give nucleoside
3 with a good 68 % yield (Scheme1). The reaction was
remarkably more efficient (83 % yield) when 7 was obtained
from the more stable hydrochloride salt of L-threonine 6a. (Note:
in a similar approach, used by Chheda and Hong for synthesis
of t6A, the “isocyanated” O-benzyl-L-threonine benzyl ester was
reacted with 2′,3′,5′-tri-O-acetyl-adenosine and the product was
obtained with a moderate 48 % yield. 50 In a “mirror” reaction of
the “isocyanated” adenosine with O-tert-butyldimethylsilyl-L-
threonine p-nitrophenylethyl ester the urethane nucleoside
product was obtained in only 19 % yield38). The acetyl groups in
3 were safely removed with 10 % TEA in methanol to give the
conjugate 4 in 86 %, so the three step synthesis (path I,
1→2→3→4, Scheme 1) afforded the TBDMS/TMSE derivative
of ms2t6A in 50 % overall yield, compared to 40 % reported for
the
use
of
O-tert-butyldimethylsilyl-L-threonine-(2-(4-
nitrophenyl)ethyl) ester intermediate.41 For the alternative two
step synthesis (path II, 1→3→4), the overall 60-70 % efficiency
was observed, depending on the way of preparation of 7, so in
terms of chemical efficiency this newly developed isocyanate
route is advantageous over the path I.
Figure 2. A) Synthesis of ms2ct6A-modified oligoribonucleotide with the
sequence corresponding to the anticodon stem and loop of tRNALys T. brucei
(ms2ct6A-ASL); B) comparison of RP-HPLC profiles of precursor ms2t6A-ASL
and cyclic ms2ct6A-ASL; C) MALDI-TOF MS for ms2ct6A-ASL
The final transformation 4→5 (Scheme 1) was
accomplished using the chemistry reported for previous
syntheses of t6A / ms2t6A phosphoramidite building blocks.38-43
The details are given in Supporting Information (Scheme S3).
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Conversion of ms2t6A → ms2ct6A at oligonucleotide level
Taking into account that the conditions applied in the synthesis
and purification of ms2ct6A-ASL might lead to the hydrolysis of
the hydantoin ring in L-ms2ct6A and/or the epimerization at the
C-α atom of the amino acid residue, four HPLC-standards were
prepared: “linear” L-ms2t6A (12a) and D-allo-ms2t6A (12b) and
“cyclic” L-ms2ct6A (13a)/ D-allo-ms2ct6A (13b) (Figure 3, part A,
and Supporting Information Scheme S4 and Figures S8, S9).
Intermediates 11a and 11b were synthesized in the reactions of
phenoxycarbamate 2 with L-threonine or its C-α epimer (D-allo-
threonine), respectively. Their treatment with 10 M methanolic
solution of ammonia afforded the corresponding ammonium
salts of 12a or 12b, which were further converted into free acids
by ion exchange chromatography and treated with the polymer-
bound 1-ethyl-3-(3-dimethyl-aminopropyl)-carbodiimide (EDC-P,
5 equiv) and hydroxybenzotriazole (HOBt, 5 equiv). After 1h
reaction at rt, the polymer beads were filtered off and “cyclic”
13a or 13b were isolated (in 90 % and 92 %, respectively) by
silica gel column chromatography. An RP-HPLC analysis
revealed that the peaks corresponding to 12a, 12b, 13a, and
13b were well separated (Figure 3, part B). Using these
standards in analysis of the aforementioned mixture of
nucleosides obtained after enzymatic hydrolysis of ms2ct6A-
ASL we confirmed the presence of the ms2ct6A unit with a
natural L-threonine residue (Figure 3, part B and C).
To convert the N6-threonylcarbamoyl residue in ms2t6A-
ASL into a hydantoin ring, we applied the conditions developed
recently for the post-synthetic t6A→ct6A conversion in t6A-
containing oligomers.33 In a small scale dehydration/cyclization
of ms2t6A-ASL (0.5 OD),
5 equivalents of 1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) and
5 equivalents of 1-hydroxy-1H-benzotriazole (HOBt) were used.
After 1 hour, an RP-HPLC analysis revealed ca. 50
conversion. Longer reaction time did not promote the further
conversion. When the excess of EDC·HCl/HOBt was doubled, 1-
hour reaction time was sufficient for almost quantitative
%
ms2t6A-ASL→ms2ct6A-ASL
Information, Figure S4). This 10-fold excess of dehydrating
reagents was maintained in large scale (50 OD)
transformation
(Supporting
a
transformation, which after ultrafiltration of the reaction mixture
(Amicon® Ultra 3K; 3,000 MWCO) furnished 45 OD (90 %
conversion) of ms2ct6A-ASL (for details see Experimental
Section and Supporting Information, Figure S5). The structure of
ms2ct6A-ASL was confirmed by MALDI-TOF MS (Figure 2,
Figure S6) and determination of the nucleoside content (Figure 3
and Figure S7). In the latter, the isolated product was hydrolyzed
with nuclease P1 and the resultant nucleoside 5’-O-phosphates
were dephosphorylated with alkaline phosphatase in 20 mM
TEA∙HCl (pH 7).21 RP-HPLC analysis (monitored at 254 nm) of
the mixture showed the presence of four canonical and one
modified nucleoside product (Figure 3, part C).
Conclusions
The optimization of the carbamate route (Scheme 1, path
I) as well as the first synthesis of the protected ms2t6A derivative
4 on the isocyanate route (path II), significantly improved the
availability of this synthetically demanding nucleoside and made
the preparation of the ms2t6A-phosphoramidite monomer 5 more
efficient. Compound 5 was used in a manual synthesis of the
17nt ms2t6A-oligonucleotide (the anticodon stem and loop
fragment of tRNALys from T. brucei), which was post-synthetically
subjected
to
the
epimerization-free
ms2t6A→ms2ct6A
cyclization. Since this approach furnishes the “linear” ms2t6A-
oligonucleotide and its “cyclic” ms2ct6A-containing derivative,
the comparative structure-activity relationship studies may
commence, which should result in a better understanding of the
role of ms2t6A cyclization in the tuning of decoding process in
protein biosynthesis.
Experimental Section
General remarks
Commercial reagents purchased from Aldrich and Acros companies were
used without additional purification unless otherwise stated. All solid
reagents were dried under high vacuum prior to use. Analytical thin layer
chromatography (TLC) was done on silica gel coated plates (60 F254,
Supelco) with UV light (254 nm) or the ninhydrin test (for amino acids)
detection. The products were purified by chromatography on a silica gel
column 60 (mesh 230 – 400, Fluka) eluted with the indicated solvent
mixtures. NMR spectra were recorded using a 700 MHz (for 1H)
instrument (176 MHz for 13C, 101 MHz for 31P). Chemical shifts (δ) are
Figure 3. A) Synthesis of L-ms2ct6A and D-allo-ms2ct6A i) L-threonine (a) or
D-allo-threonine (b) (3.0 equiv), pyridine, 40 °C, 10 h, 88 % yield for 11a, 82 %
yield for 11b; ii) NH3 in methanol (10 M), rt, 5 h, 90 % yield for 12a; 87 % yield
for 12b; iii) EDC-P (5 equiv), HOBt (5 equiv), water, rt, 1 h, 90 % yield for 13a,
92 % yield for 13b; B) RP-HPLC profile of a mixture of L-ms2t6A / D-allo-
ms2t6A and L-ms2ct6A / D-allo-ms2ct6A nucleosides C) The nucleoside
composition analysis of ms2ct6A-ASL after enzymatic digestion.
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reported in ppm relative to TMS (an internal standard) for 1H and 13C,
and 85 % phosphoric acid (an external standard) for 31P. The signal
multiplicities are described as s (singlet), d (doublet), dd (doublet of
doublets), dt (doublet of triplets), dq (doublet of quartets), t (triplet), td
(triplet of doublets), q (quartet), qd (quartet of doublets), m (multiplet),
and bs (broad singlet). MALDI-TOF MS spectra were recorded on
CH3-CO), 1.19 (d, 3H,3J = 6.2 Hz, CH3 Thr), 1.03-0.94 (m, 2H, Si-CH2 TMSE),
0.85 (s, 9H, Si-C(CH3)3 TBDMS), 0.07 (s, 3H, Si-CH3 TBDMS), 0.03 (s, 3H,
Si-CH3 TBDMS), 0.01 (s, 9H, Si(CH3)3 TMSE); 13C NMR: (DMSO-d6) δ:
170.25 (C=O Thr), 169.96 (CH3-CO), 169.43 (CH3-CO), 169.30 (CH3-CO),
164.58 (C-2), 153.23 (NH-CO-NH), 150.68 (C-4), 149.90 (C-6), 141.81 (C-8),
118.03 (C-5), 86.50 (C-1'), 78.88 (C4'), 71.96 (C-2'), 69.60 (C-3'), 68.69 (CH-β
Thr), 62.89 (O-CH2 TMSE), 62.51 (C-5′), 59.04 (CH-α Thr), 25.71 (Si-C(CH3)3
TBDMS), 20.56 (CH3 Thr), 20.35 (CH3-CO), 20.33 (CH3-CO), 20.26 (CH3-CO),
17.71 (Si-C(CH3)3 TBDMS), 16.79 (Si-CH2 TMSE), 14.12 (S-CH3), -1.58
(Si(CH3)3 TMSE), -4.25 (Si-CH3 TBDMS), -5.26 (Si-CH3 TBDMS) (Spectra
S16/S17, Supporting Information).
Applied Biosystems Voyager-Elite mass spectrometer in
a linear,
negative-ion mode. IR data were recorded on an FT-IR ALPHA
instrument (Bruker) equipped with a Platinum ATR QuickSnap™ module.
Analytical HPLC of nucleosides/oligonucleotides were performed on a
Shimadzu Prominence HPLC system equipped with an SPD-M20A
spectral photodiode array detector using a Kinetex® column (RP, C18, 5
µm, 4.6 × 250 mm, 100 Å, Phenomenex) or an Ascentis® column (RP,
N-[[9-(β-ᴅ-ribofuranosyl)-2-methylthiopurin-6-yl]carbamoyl]-O-tert-
butyldimethylsilyl-ʟ-threonine trimethylsilylethyl ester (4)
C18,
5 µm, 4.6 × 250 mm, Supelco). Preparative IEX-HPLC of
To a solution of 3 (0.98 g; 1.3 mmol) in anhydrous MeOH (5 mL) a 10 %
solution of TEA in MeOH (5 mL) was added and allowed to stir at room
temperature. After 24 h reaction mixture was diluted with toluene (50 mL)
and the solvents were removed under reduced pressure. The oily residue
was extra co-evaporated with toluene (3 × 15 mL) and the product 4 was
isolated by column chromatography (silica gel, 0-7 % MeOH in CHCl3) as
a white solid (0.75 g; 1.1 mmol; 86 % yield). TLC: Rf = 0.18 (CHCl3/MeOH,
95/5, v/v); 1H NMR: (DMSO-d6) δ: 9.90 (s, 1H, NH-6), 9.27 (d, 1H, 3J = 8.5
Hz, NH Thr), 8.51 (s, 1H, H-8), 5.93 (d, 1H, 3J = 5.7 Hz, H-1'), 5.49 (d, 1H,
3J = 6.0 Hz, 2'-OH), 5.22 (d, 1H, 3J = 5.0 Hz, 3'-OH), 4.99 (t, 1H, 3J = 5.6 Hz,
5'-OH), 4.64-4.62 (m, 1H, H-2'), 4.47-4.43 (m, 2H, CH-α Thr, CH-β Thr),
oligoribonucleotides were performed on a Waters 515 HPLC system
equipped with a 996 spectral diode array detector, using a SOURCE™
column (IE, 15Q 4.6/100PE®, GE Healtcare). Analyses were run at 30 °C
for RP-HPLC and at 38 °C for IEX-HPLC. The elution profiles were UV
monitored at λ = 254 nm.
Synthesis of 2-methylthio-N6-threonylcarbamoyladenosine protected
at threonyl –OH and –COOH functions (4)
[9-(2',3',5'-tri-O-acetyl-β-ᴅ-ribofuranosyl)-2-methylthiopurin-6-yl]
phenylcarbamate (2)
2
3
To a sample of 2',3',5'-tri-O-acetyl-2-methylthioadenosine (1) (0.60 g;
1.4 mmol) dissolved in anhydrous 1,4-dioxane (3 mL) phenoxycarbonyl
tetrazole (0.80 g; 4.2 mmol) was added in one portion under argon
atmosphere. The mixture was stirred at 40 °C for 20 h and the solvent
was evaporated under reduced pressure. The product 2 was isolated by
column chromatography (silica gel, 0-20 % AcOEt in CH2Cl2) as a white
solid (0.62 g; 1,1 mmol; 78 % yield). TLC: Rf = 0.74 (CHCl3/MeOH, 95/5,
v/v); 1H NMR: (DMSO-d6) δ: 11.19 (s, 1H, NH-6), 8.51 (s, 1H, H-8), 7.47-7.43
(m, 2H, HAr), 7.30-7.27 (m, 1H, HAr), 7.26-7.25 (m, 2H, HAr), 6.26 (d,1H, 3J =
4.4 Hz, H-1'), 6.07 (dd, 1H, 3J = 6.2 Hz, 3J = 4.4 Hz, H-2'), 5.70-5.68 (m, 1H,
H-3'), 4.42 (dd, 1H, 2J = 12.0 Hz, 3J = 3.7 Hz, H-5'), 4.39-4.37 (m, 1H, H-4'),
4.22 (dd, 1H, 2J = 12.0 Hz, 3J = 5.4 Hz, H-5″), 2.59 (s, 3H, S-CH3), 2.12 (s, 3H,
CH3-CO), 2.07 (s, 3H, CH3-CO), 1.96 (s, 3H, CH3-CO). (Spectrum S15,
Supporting Information).
4.22-4.16 (m, 2H, H-3', O-CH2 TMSE), 4.11 (td, 1H, J = 10.8 Hz, J = 6.1
Hz, O-CH2 TMSE), 3.95-3.94 (m, 1H, H-4'), 3.68-3.65 (m, 1H, H-5'), 3.60-
3.53 (m, 1H, H-5″), 2.56 (s, 3H, S-CH3), 1.19 (d, 3H, 3J = 6.1 Hz, CH3 Thr),
1.03-0.94 (m, 2H, Si-CH2 TMSE), 0.85 (s, 9H, Si-C(CH3)3 TBDMS), 0.08 (s,
3H, Si-CH3 TBDMS), 0.03 (s, 3H, Si-CH3 TBDMS), 0.01 (s, 9H, Si(CH3)3
TMSE); 13C NMR: (DMSO-d6) δ: 170.26 (C=O Thr), 163.95 (C-2), 153.30
(NH-CO-NH), 151.26 (C-4), 149.70 (C-6), 141.24 (C-8), 117.80 (C-5), 87.46
(C-1'), 85.54 (C4'), 73.53 (C-2'), 70.31 (C-3'), 68.72 (CH-β Thr), 62.87
(O-CH2 TMSE), 61.34 (C-5'), 59.02 (CH-α Thr), 25.71 (Si-C(CH3)3 TBDMS),
20.54 (CH3 Thr), 17.71 (Si-C(CH3)3 TBDMS), 16.79 (Si-CH2 TMSE), 14.13
(S-CH3), -1.59 (Si-(CH3)3 TMSE), -4.30 (Si-CH3 TBDMS), -5.23 (Si-CH3
TBDMS). (Spectra S18/S19, Supporting Information).
Synthesis of L-threonine components
N-(tert-butoxycarbonyl)-O-tert-butyldimethylsilyl-ʟ-threonine (9)
N-[[9-(2',3',5'-tri-O-acetyl-β-ᴅ-ribofuranosyl)-2-methylthiopurin-6-yl]carbamoyl]-
O-tert-butyldimethylsilyl-ʟ-threonine trimethylsilylethyl ester (3)
N-tert-butoxycarbonyl-ʟ-threonine
8
(5.00 g; 22.8 mmol) was
co-evaporated with anhydrous pyridine (3 × 15 mL), dissolved in dry
pyridine (40 mL) and treated with imidazole (3.42 g; 50 mmol) and
TBDMS-Cl (7.54 g; 50 mmol). After stirring for 72 h at room temperature,
H2O (150 mL) was added and the stirring was continued for 0.5 h. The
mixture was extracted with AcOEt (3 × 150 mL). The combined organic
layers were washed with 1M aq. KHSO4 (2 × 150 mL) and H2O (150 mL),
dried over anhydrous MgSO4, filtered and evaporated under reduced
pressure. The oily residue was co-evaporated with toluene (3 × 150 mL)
and the product 9 was isolated by column chromatography (silica gel,
0-10 % MeOH in CH2Cl2) as colorless oil (7.45 g; 22.3 mmol, 98 % yield).
Path I: To a solution of 2 (1.12 g; 2.0 mmol) in dry pyridine (9 mL)
threonine trifluoroacetate 6 (1.00 g; 2.2 mmol) was added. The mixture
was stirred at 40 °C for 24 h and the solvent was evaporated under
reduced pressure. The oily residue was co-evaporated with toluene (3 ×
15 mL), diluted with CH2Cl2 and washed with 5 % aq. NaHCO3 (2 × 40
mL) and H2O (50 mL). The organic layer was dried over anhydrous
MgSO4, filtered and the solvent was evaporated under reduced pressure.
The product 3 was isolated by column chromatography (silica gel, 0-1 %
MeOH in CH3Cl) as a light-yellow solid (1.13 g; 1.5 mmol; 75 % yield).
1
TLC: Rf = 0.33 (AcOEt/hexane/AcOH, 60/40/1, v/v); H NMR: (DMSO-d6)
Path II: 2',3',5'-Tri-O-acetyl-2-methylthioadenosine (1, 0.31 g; 0.70 mmol)
and threonine isocyanate (crude 7 obtained from 6a, 0.76 g; 2.10 mmol)
were dissolved in dry toluene (12 mL). The reaction mixture was stirred
under reflux for 16 h and the solvent was evaporated under reduced
pressure. The product 3 was isolated by column chromatography (silica
gel, 0-1 % MeOH in CHCl3) as a light-yellow solid (0.47 g; 0.58 mmol;
83 % yield).
TLC: Rf = 0.52 (CHCl3/MeOH, 95/5, v/v); 1H NMR: (DMSO-d6) δ: 9.98 (s,1H,
NH-6), 9.25 (d, 1H, 3J = 8.6 Hz, NH Thr), 8.45 (s, 1H, H-8), 6.25 (d, 1H,3J =
4.3 Hz, H-1'), 6.08 (dd, 1H,3J = 6.0 Hz, 3J = 4.3 Hz, H-2'), 5.70-5.68 (m, 1H,
H-3'), 4.46-4.44 (m, 2H, CH-α Thr, CH-β Thr), 4.42 (dd, 1H, 2J = 12.0 Hz, 3J =
3.7 Hz, H-5'), 4.38 (dt, 1H, 3J = 5.8 Hz, 3J = 3.8 Hz, H-4'), 4.22-4.18 (m, 2H, H-
5″,O-CH2 TMSE), 4.11 (dt, 1H, 2J = 10.7 Hz, 3J = 6.2 Hz, O-CH2 TMSE), 2.58
(s, 3H, S-CH3), 2.11 (s, 3H, CH3-CO), 2.08 (s, 3H, CH3-CO), 1.96 (s, 3H,
δ: 12.71 (s, 1H, COOH), 5.91 (d, 1H, 3J = 9.8 Hz, NH), 4.30 (dq, 1H, 3J =
3
3
3
6.2 Hz, J = 2.8 Hz, CH-β), 3.97 (dd, 1H, J = 9.1 Hz, J = 2.1 Hz, CH-α),
3
1.39 (s, 9H, C(CH3)3 Boc), 1.11 (d, 3H, J = 6.3 Hz, CH3), 0.82 (s, 9H, Si-
C(CH3)3 TBDMS), 0.03 (s, 3H, Si-CH3 TBDMS), 0.00 (s, 3H, Si-CH3
TBDMS); 13C NMR: (DMSO-d6) δ: 172.09 (C=O), 156.56 (C=O Boc), 78.40
(C(CH3)3 Boc), 68.39 (CH-β), 59.13 (CH-α), 28.09 (C(CH3)3 Boc), 25.63 (Si-
C(CH3)3 TBDMS), 20.55 (CH3), 17.62 (Si-C(CH3)3 TBDMS), -4.54 (Si-CH3
TBDMS), -5.25 (Si-CH3 TBDMS). (Spectra S5/S6, Supporting Information).
N-(tert-butoxycarbonyl)-O-tert-butyldimethylsilyl-ʟ-threonine
trimethylsilylethyl ester (10)
To a solution of protected threonine 9 (3.00 g; 9.0 mmol) in anhydrous
CH2Cl2 (40 mL) 2-(trimethylsilyl)ethanol (1.81 mL; 12 mmol) was added.
The mixture was stirred for 10 minutes at 0 °C and DCC (3.10 g;
This article is protected by copyright. All rights reserved.

10.1002/chem.201902411
Chemistry - A European Journal
FULL PAPER
15 mmol) and DMAP (0.11 g, 0.9 mmol) were added. After stirring for 3 h,
the DCU precipitate was filtered off and washed with CH2Cl2 (3 × 10 mL).
The solvent was evaporated under reduced pressure and the product 10
was isolated by column chromatography (silica gel, hexane/CHCl3, 1:1,
v/v) as a white solid (3.82 g; 8.8 mmol; 98 % yield). TLC: Rf = 0.45
(CHCl3); 1H NMR: (DMSO-d6) δ: 6.19 (d, 1H, 3J = 9.4 Hz, NH), 4.27 (dq,
0.26 for 6a in CHCl3/MeOH, 98/2, v/v). The mixture was diluted with
CH2Cl2 (24 mL) and poured into a mixture of 0.5 M aq. HCl (24 mL) and
crushed ice (24 mL). The organic layer was collected and the above
wash step was repeated. The combined aqueous layers were extracted
with CH2Cl2 (40 mL). The organic layers were combined and washed with
brine (30 mL), water (30 mL) and dried over anhydrous MgSO4. The
solvents were removed under reduced pressure to afford 7 as a light
brown oil. The crude product, without further purification, was used in the
reaction with 1. TLC: Rf = 0.94 (CHCl3/MeOH, 98/2, v/v); IR (film) 2245 cm-1
(N=C=O), Figure S1, Supporting Information ; 1H NMR: (CDCl3) δ: 4.36 (dq,
1H, 3J = 6.2 Hz, 3J = 1.9 Hz, CH-β), 4.29 (dt, 1H, 2J = 10.9 Hz, 3J = 6.8 Hz, O-
CH2 TMSE), 4.21 (dt, 1H, 2J = 10.8 Hz, 3J = 6.7 Hz, O-CH2 TMSE), 3.62 (d,
1H, 3J = 1.9 Hz, CH-α), 1.26 (d, 3H, 3J = 6.3 Hz, CH3), 1.08-1.00 (m, 2H,
Si-CH2 TMSE), 0.87 (s, 9H, Si-C(CH3)3 TBDMS), 0.07 (s, 3H, Si-CH3
TBDMS), 0.05 (s, 9H, Si(CH3)3 TMSE), 0.00 (s, 3H, Si-CH3 TBDMS); 13C
NMR: (DMSO-d6) δ: 170.06 (C=O), 128.98 (N=C=O), 69.34 (CH-β), 64.89
(O-CH2 TMSE), 64.67 (CH-α), 25.62 (Si-C(CH3)3 TBDMS), 21.33 (CH3),
17.95 (Si-C(CH3)3 TBDMS), 17.54 (Si-CH2 TMSE), -1.46 (Si(CH3)3
TMSE), -4.07 (Si-CH3 TBDMS), -5.16 (Si-CH3 TBDMS). (Spectra S13/S14,
Supporting Information).
3
3
2
3
1H, J = 6.2 Hz, J = 3.1 Hz, CH-β), 4.14 (dt, 1H, J = 10.7 Hz, J = 6.5
Hz, O-CH2 TMSE), 4.09 (dt, 1H, 2J = 10.7 Hz, 3J = 6.2 Hz, O-CH2 TMSE),
3
4.04 (dd, 1H, 3J = 9.4 Hz, J = 3.1 Hz, CH-α), 1.39 (s, 9H, C(CH3)3 Boc),
3
1.11 (d, 3H, J = 6.2 Hz, CH3), 0.99-0.90 (m, 2H, Si-CH2 TMSE), 0.82 (s,
9H, Si-C(CH3)3 TBDMS), 0.03 (s, 12H, Si(CH3)3 TMSE; Si-CH3
TBDMS), -0.01 (s, 3H, Si-CH3 TBDMS); 13C NMR: (DMSO-d6) δ: 170.62
(C=O), 155.52 (C=O, Boc), 78.52 (C(CH3)3 Boc), 68.35 (CH-β), 62.66
(O-CH2 TMSE), 59.42 (CH-α), 28.07 (C(CH3)3 Boc), 25.53 (Si-C(CH3)3
TBDMS), 20.36 (CH3), 17.55 (Si-C(CH3)3 TBDMS), 16.76 (Si-CH2 TMSE),
-1.58 (Si-(CH3)3 TMSE), -4.44 (Si-CH3 TBDMS), -5.42 (Si-CH3 TBDMS).
(Spectra S7/S8, Supporting Information).
Trifluoroacetate of O-tert-butyldimethylsilyl-ʟ-threonine trimethylsilylethyl
ester (6)
To a solution of N-Boc trimethylsilylethyl ester 10 (3.00 g; 6.9 mmol) in
CH2Cl2 (3.3 mL) was treated with freshly prepared mixture of TFA in
anhydrous CH2Cl2 (60 vol %, 16 mL). After 5 min stirring at room
temperature, toluene (9 mL) was added and the solvents were
evaporated under reduced pressure. The residue was dissolved in CHCl3
(70 mL) and washed with H2O (70 mL), 4 % aq. NaHCO3 (2 × 70 mL),
and H2O (70 mL). The organic layer was dried over anhydrous MgSO4,
filtered and concentrated under reduced pressure. The product 6 was
isolated by column chromatography (silica gel, 0-1 % MeOH in CHCl3) as
Synthesis of the (ms2t6A-ASL) oligonucleotide.
The manual synthesis was performed (according to the protocol reported
by Sproat35) at a 2.5 μmol scale using 71.4 mg of the rG-succinyl-CPG
(Proligo) support (the loading of 35 µmol/g) The commercially available
phosphoramidite derivatives of A, C, U and G, carrying standard 5’-O-
DMTr and 2’-O-TBDMS protecting groups and 4-tert-butylphenoxyacetyl
(tac) moiety on the exocyclic amine functions in A, C, and G, were used
as 0.1 M solutions in anhydrous acetonitrile. The coupling reactions were
promoted with a 0.25 M solution of 5-(3,5-bis(trifluoromethyl)phenyl)-1H-
tetrazole (Activator 42) in ACN. The coupling steps with the canonical A,
U, C and G amidities were performed once (for 8 min), while for the
modified unit double coupling was executed, each for 25 minutes. The
capping steps were performed with tac anhydride (Fast deprotection
Cap A : Cap B 1:1.1 v/v) for 2 min. A 0.02 M solution of elemental iodine
in THF-H2O-pyridine (90.54:9.05:0.41 v/v/v) was used as an oxidizing
agent (2 min contact) for each oxidation step, followed by an additional
capping step (to remove traces of iodine) and washing with dry
acetonitrile. After the last coupling, the DMTr group was removed and the
support was washed with acetonitrile, dried with argon and transferred to
a screw cap glass vial for deprotection steps. To remove the 2-
cyanoethyl protecting groups the support carrying already detritylated
oligomer was suspended in a TEA-ACN mixture (3.3 mL, 1:1 v/v) and the
suspension was stirred for 20 min in a tightly closed vessel. The liquid
phase was removed and the CPG sediment was washed with ACN (3 ×
2.5 mL), dried in vacuo for 30 min and treated with 8 M ethanolic
ammonia (4.5 mL) for 16 h at 37 °C to remove the base-labile protecting
groups and cleave the ms2t6A-RNA oligonucleotide from the solid
support. The supernatant was collected and the CPG-support was rinsed
with ethanol (3 × 4 mL). The ethanolic solutions were combined and the
solvent was evaporated using a Speed-Vac concentrator. The solid
residue was dried under high vacuum for 2 h and treated with 1 M TBAF
in THF (1.8 mL) for 24 h at room temperature. The reaction was
quenched by addition of phosphate buffer (0.2 M, 5 mL, pH 7) and
desalted on a Sephadex column (G-25) using 20 % EtOHaq as eluent.
The fully deprotected oligomer was purified by high resolution anion
exchange (AEX) HPLC (a Source 15Q 4.6/100PE column) using linear
gradient at a flow rate of 1 mL min-1 (Figure S2). The column was eluted
with a linear gradient 50 mM to 600 mM NaBr in 20 mM Na2HPO4-
NaH2PO4 buffer (pH 7.5) containing 50 μM EDTA and 10 % ACN. The
ms2t6A-ASL oligomer was eluted at Rt = 31.84 min and partially
evaporated on Speed-Vac concentrator to remove acetonitrile. The
remaining solution was diluted with 100 mM AcONa (up to ~ 6 mL) and
loaded slowly onto a Sep-Pak C18 cartridge (Waters) equilibrated with
a
white solid (1.95 g; 4.5 mmol; 64
%
yield). TLC: Rf = 0.30
1
3
(CHCl3/MeOH, 98/2, v/v); H NMR: (DMSO-d6) δ: 4.16 (dq, 1H, J = 6.3
Hz, J = 2.9 Hz, CH-β), 4.14-4.11 (m, 1H, O-CH2 TMSE), 4.07-4.04 (m,
1H, O-CH2 TMSE), 3.17 (d, 1H, J = 2.8 Hz, CH-α), 1.59 (bs, 3H, NH3+),
3
3
1.16 (d, 3H, 3J = 6.3 Hz, CH3), 0.99-0.92 (m, 2H, Si-CH2 TMSE), 0.81 (s,
9H, Si-C(CH3)3 TBDMS), 0.03 (s, 9H, Si(CH3)3 TMSE), 0.02 (s, 3H, Si-
CH3 TBDMS), -0.03 (s, 3H, Si-CH3 TBDMS); 13C NMR: (DMSO-d6) δ:
174.19 (C=O), 69.67 (CH-β), 61.98 (O-CH2 TMSE), 59.90 (CH-α), 25.53
(Si-C(CH3)3 TBDMS), 20.35 (CH3), 17.57 (Si-C(CH3)3 TBDMS), 16.83
(Si-CH2 TMSE), -1.57 (Si(CH3)3 TMSE), -4.36 (Si-CH3 TBDMS), -5.35
(Si-CH3 TBDMS). (Spectra S9/S10, Supporting Information).
Hydrochloride of O-tert-butyldimethylsilyl-ʟ-threonine trimethylsilylethyl
ester (6a)
To a solution of threonine trifluoroacetate 6 (1.14 g; 2.54 mmol) in
anhydrous CH2Cl2 (10.8 mL), anhydrous 1,4-dioxane saturated with
hydrogen chloride (4 M, 1.1 mL) was added dropwise. After 5 min stirring
the solution was concentrated under reduced pressure and the oily
residue was co-evaporated with anhydrous toluene (2 × 20 mL). The
product 6a was obtained as a white solid (0.94 g; 2.54 mmol; 100 %
1
yield). TLC: Rf = 0.26 (CHCl3/MeOH, 98/2, v/v); H NMR: (CDCl3) δ: 8.71
(bs, 3H, NH3+), 4.50 (dq, 1H , 3J = 6.6 Hz, 3J = 1.7 Hz, CH-β), 4.30-4.26 (m,
3
1H, O-CH2 TMSE), 4.25-4.20 (m, 1H, O-CH2 TMSE), 4.03 (d, 1H, J = 1.7
Hz, CH-α), 1.49 (d, 3H, 3J = 6.6 Hz, CH3), 1.08-1.03 (m, 2H, Si-CH2 TMSE),
0.85 (s, 9H, C-Si(CH3)3 TBDMS), 0.08 (s, 3H, Si-CH3 TBDMS), 0.03 (s, 9H,
Si(CH3)3 TMSE), 0.02 (s, 3H, Si-CH3 TBDMS); 13C NMR: (DMSO-d6) δ:
167.77 (C=O), 67.58 (CH-β), 65.34 (O-CH2 TMSE), 58.86 (CH-α), 25.83
(Si-C(CH3)3 TBDMS), 21.56 (CH3), 18.02 (Si-C(CH3)3 TBDMS), 17.44 (Si-
CH2 TMSE), -1.45 (Si(CH3)3 TMSE), -4.08 (Si-CH3 TBDMS), -5.09 (Si-CH3
TBDMS). (Spectra S11/S12, Supporting Information).
“Isocyanated” O-tert-butyldimethylsilyl-ʟ-threonine trimethylsilylethyl ester (7)
A solution of 6a (0.94 g, 2.54 mmol) in anhydrous CH2Cl2 (8.6 mL) and
anhydrous pyridine (0.9 mL) was stirred under argon atmosphere in an
ice bath for 15 min. Then, a 20 % solution of phosgene in toluene (2 mL)
was added dropwise and the resultant yellow solution was stirred at 0 °C
for 2 h (TLC analysis revealed 100 % conversion; Rf = 0.94 for 7 vs. Rf =
This article is protected by copyright. All rights reserved.

10.1002/chem.201902411
Chemistry - A European Journal
FULL PAPER
100 mM AcONa (pH 6.5, 10 mL). The packing bed was drop-by-drop
washed with H2O (miliQ quality, 6 × 8 mL). Finally, the cartridge was
flushed with the elution solution (ACN/H2O, 9:1, v/v) and the collected
fraction was evaporated to dryness and lyophilized to give 94 OD260 units
of precursor ms2t6A-ASL. The obtained product was analyzed by
MALDI-TOF mass spectrometry (Figure S3).
(CH3-CO), 169.41 (CH3-CO), 169.29 (CH3-CO), 164.36 (C-2), 153.02
(NH-CO-NH), 150.66 (C-4), 149.98 (C-6), 141.71 (C-8), 117.97 (C-5),
86.38 (C-1'), 78.96 (C-4'), 71.96 (C-2'), 69.64 (C-3'), 66.57 (CH-β Thr),
62.49 (C-5'), 59.19 (CH-α Thr), 20.37 (CH3-CO), 20.31 (CH3-CO), 20.22
(CH3-CO), 19.02 (CH3 Thr), 13.96 (S-CH3). (Spectra S27/S28,
Supporting Information).
2-Thiomethyl-ʟ-N6-threonylcarbamoyladenosine (12a) and 2-thiomethyl-
ᴅ-allo-N6-threonylcarbamoyladenosine (12b)
Cyclization of ms2t6A to ms2ct6A at the oligonucleotide level
Compound 11a (0.21 g; 0.36 mmol) was dissolved in anhydrous MeOH
saturated with NH3 (10 M, 5 mL) and the mixture was stirred at room
temperature for 5 h. The solvent was evaporated and the residue was
dissolved in water and lyophilized. The resultant ammonium salt was
then converted into an acidic form by ion exchange chromatography on
Amberlite™ IR120 (H+ form). The column was eluted with water and the
eluate was concentrated down to 5 mL and lyophilized to yield 12a as a
white solid (0.15 g; 0.33 mmol; 90 % yield). Rf = 0.44 (n-BuOH/H2O/AcOH,
5:3:2 v/v/v); 1H NMR: (DMSO-d6) δ: 9.74 (bs, 1H, NH-6), 9.35 (d, 1H,
3J = 8.4 Hz, NH Thr), 8.51 (s, 1H, H-8), 5.91 (d, 1H, 3J = 6.3 Hz, H-1'), 5.49
(bs, 1H, 2'-OH), 5.21 (bs, 1H, 3'-OH), 5.01 (bs, 1H, 5'-OH), 4.58-4.59 (m,
1H, H-2'), 4.30 (dd, 1H, 3J = 9.1 Hz, 3J = 2.8 Hz, CH-α Thr), 4.26-4.23 (m,
1H, CH-β Thr), 4.17-4.16 (m, 1H, H-3'), 3.94 (dd, 1H, 3J = 8.4 Hz, 3J = 4.2
Hz, H-4'), 3.67-3.65 (m, 1H, H-5'), 3.57-3.55 (m, 1H, H-5″), 2.58 (s, 3H, S-
CH3), 1.13 (d, 3H, 3J = 6.3 Hz, CH3 Thr); 13C NMR (DMSO-d6) δ: 172.25
(C=O Thr), 164.06 (C-2), 153.33 (NH-CO-NH), 151.24 (C-4), 149.84 (C-6),
141.17 (C-8), 117.81 (C-5), 87.32 (C-1'), 85.57 (C-4'), 73.55 (C-2'), 70.30
(C-3'), 66.26 (CH-β Thr), 61.31 (C-5'), 58.51 (CH-α Thr), 20.67 (CH3 Thr),
14.06 (S-CH3). (Spectra S29/S30, Supporting Information).
To the lyophilized 5’-CACGGCUUUUms2t6AACCGUG-3’ (50 OD, n =
3.6·10-4 mmol, m = 2.0 mg) the 100 µL aliquots of the freshly prepared
solutions of 7.0 mg of EDC·HCl in 1 mL H2O (10 equiv, n = 3.6·10-3 mmol,
m = 0.70 mg) and 4.8 mg HOBt in 1mL DMF (10 equiv, n = 3.6·10-3 mmol,
m = 0.48 mg) were added. An HPLC analysis performed after 1 hour
incubation at 25 °C showed one main product (Rt = 14.73 min, Supporting
Information, Figure S5). The oligomer was isolated by ultrafiltration using
an Amicon® Ultra-4 Centrifugal Filter 3.000 MWCO (for volumes up to 4
mL). The crude sample was placed in the filter device and diluted to 4 mL
with 200 mM NaCl. The filter was put in a fixed-angle rotor and a 7,500×g
acceleration was applied for 40 min. The filtrate was removed and the
filtration was repeated three times using miliQ quality H2O. Finally, the
oligonucleotide was transferred from device to an Eppendorf tube and
evaporated to dryness affording 45 OD260 units (90 % yield). The
obtained ms2ct6A-ASL oligonucleotide was analyzed by MALDI-TOF
mass spectrometry (m/z calc. 5535, found 5537) and its homogeneity
was confirmed by RP-HPLC (Figures S5, S6, Supporting Information).
Synthesis of L-ms2t6A (12a) / D-allo-ms2t6A (12b) and L-ms2ct6A (13a)
/ D-allo-ms2ct6A (13b) nucleosides
Compound 12b was prepared analogously as 12a starting from 11b and
was obtained as a white solid (0.14 g; 0.31 mmol; 87% yield). Rf = 0.44
(n-BuOH/H2O/AcOH, 5:3:2 v/v/v); 1H NMR: (DMSO-d6) δ: 9.81 (bs, 1H,
NH-6), 9.45 (d, 1H, 3J = 8.4 Hz, NH Thr), 8.52 (s, 1H, H-8), 5.93 (d, 1H,
3J = 6.3 Hz, H-1'), 5.51 (bs, 1H, 2'-OH), 5.23 (bs, 1H, 3'-OH), 5.00 (bs, 1H,
5'-OH), 4.62-4.61 (m, 1H, H-2′), 4.38 (dd, 1H, 3J = 8.4 Hz, 3J = 4.9 Hz, CH-α
Thr), 4.18-4.17 (m, 1H, H-3'), 4.06-4.05 (m, 1H, CH-β Thr), 3.69-3.95 (m,
1H, H-4'), 3.68-3.65 (m, 1H, H-5'), 3.56-3.55 (m, 1H, H-5″), 2.59 (s, 3H,
S-CH3), 1.19 (d, 3H, 3J = 6.3 Hz, CH3 Thr); 13C NMR (DMSO-d6) δ: 171.82
(C=O Thr), 163.80 (C-2), 153.11 (NH-CO-NH), 151.27 (C-4), 149.82 (C-6),
141.19 (C-8), 117.76 (C-5), 87.39 (C-1'), 85.57 (C-4'), 73.58 (C-2'), 70.33
(C-3'), 66.62 (CH-β Thr), 61.35 (C-5'), 59.15 (CH-α Thr), 19.13 (CH3 Thr),
13.94 (S-CH3). (Spectra S31/S32, Supporting Information).
2′,3′,5′-Tri-O-acetyl-2-thiomethyl-ʟ-N6-threonylocarbamoyladenosine
(11a) and 2′,3′,5′-tri-O-acetyl-2-thiomethyl-ᴅ-allo-N6-threonylocarbamoyl-
adenosine (11b)
To the solution of 2 (0.67 g; 1.2 mmol) in anhydrous pyridine (5 mL)
ʟ-threonine (0.43 g, 3.6 mmol) was added. The mixture was stirred at
40 oC for 10 h and excess of amino acid was filtered off and washed with
pyridine. The filtrates were combined and the solvent was evaporated
under reduced pressure. The residue was co-evaporated with toluene (3 x
5 mL) and the product 11a was isolated by column chromatography (silica
gel, CH2Cl2/MeOH, 7:3, v/v) as a white solid (0.59 g; 1.0 mmol; 88 % yield).
1
Rf = 0.67 (n-BuOH/H2O/AcOH, 5:3:2, v/v/v); H NMR: (DMSO-d6) δ: 9.84
2-thiomethyl-ʟ-N6-threonylcarbamoyladenosine
(13a)
and
(bs, 1H, NH-6), 9.32 (d, 1H, 3J = 8.4 Hz, NH Thr), 8.46 (s, 1H, H-8), 6.23 (d,
1H, 3J = 4.9 Hz, H-1'), 6.02 (dd, 1H, 3J = 5.6 Hz, 3J = 4.9 Hz, H-2'),
5.67-5.65 (m, 1H, H-3'), 4,42-4,37 (m, 2H, H-4', H-5'), 4.28 (dd, 1H, 3J = 8.4
Hz, 3J = 2.1 Hz, CH-α Thr) 4.25-4.22 (m, 2H, H-5″, CH-β Thr), 2.59 (s, 3H,
S-CH3), 2.12 (s, 3H, CH3-CO), 2.06 (s, 3H, CH3-CO), 1.98 (s, 3H, CH3-CO),
1.13 (d, 3H, 3J = 6.3 Hz, CH3 Thr); 13C NMR (DMSO-d6) δ: 172.19 (C=O
Thr), 169.97 (CH3-CO), 169.39 (CH3-CO), 169.25 (CH3-CO), 164.62 (C-2),
153.24 (NH-CO-NH), 150.74 (C-4), 150.00 (C-6), 141.59 (C-8), 117.96 (C-
5), 86.05 (C-1'), 79.15 (C-4'), 71.98 (C-2'), 69.75 (C-3'), 66.22 (CH-β Thr),
62.61 (C-5'), 58.53 (CH-α Thr), 20.65 (CH3 Thr), 20.40 (CH3-CO), 20.32
(CH3-CO), 20.21 (CH3-CO), 14.04 (S-CH3). (Spectra S25/S26, Supporting
Information).
Cyclic
2-thiomethyl-ᴅ-allo-N6-threonylcarbamoyladenosine (13b)
To the solution of 12a (50 mg; 0.11 mmol) in Milli-Q® water (5 mL) the
polymer-bound EDC was added (390 mg; 0.55 mmol). The suspension
was stirred at room temperature for 5 min and HOBt (74 mg; 0.55 mmol)
was added. After 1 h stirring, the resin was filtered off and washed with
H2O (3 x 5 mL), MeOH (2 x 5 mL) and the collected solvents were
removed under reduced pressure. The product 13a was isolated by
column chromatography (silica gel, 0-2 % H2O in n-BuOH) as a white
solid (43 mg; 0.10 mmol; 90% yield). Rf = 0,.48 (n-BuOH/H2O, 85:15, v/v);
1H NMR: (DMSO-d6) δ: 8.72 (s, 1H, H-8), 8.71 (s, 1H, NH Thr), 6.00 (d,
3J = 5.7 Hz, H-1'), 5.57 (d, 1H, 3J = 5.9 Hz, 2'-OH), 5.24 (d, 1H, 3J = 4.9 Hz,
3'-OH), 5.20 (d, 1H, 3J = 5.9 Hz, -OH Thr), 5.01 (t, 1H, 3J = 5.6 Hz, 5'-OH),
4.71-4.69 (m, 1H, H-2'), 4.22 (dd, 1H, 3J = 2.7 Hz, 3J = 1.2 Hz, CH-α Thr),
4.22-4.20 (m, 1H, H-3'), 4.08-4.03 (m, 1H, CH-β Thr), 3.97-3.96 (m, 1H,
H-4'), 3.68-3.65 (m, 1H, H-5'), 3.59-3.55 (m, 1H, H-5″), 2.61 (s, 1H, S-CH3),
Compound 11b was prepared analogously as 11a starting from 2 and
ᴅ-allo-threonine and it was obtained as a white solid (0.56 g; 0.95 mmol;
82 % yield). Rf = 0.65 (n-BuOH/H2O/AcOH, 5:3:2 v/v/v); 1H NMR:
(DMSO-d6) δ: 9.89 (bs, 1H, NH-6), 9.42 (d, 1H, 3J = 7.7 Hz, NH Thr),
8.45 (s, 1H, H-8), 6.24 (d, 1H, 3J = 4.2 Hz, H-1'), 6.06-6.05 (m, 1H, H-2'),
5.69-5.68 (m, 1H, H-3'), 4.42 (dd, 1H, 2J = 12.6 Hz, 3J = 4.2 Hz, H-5'),
4.39-4.35 (m, 2H, H-4', CH-α Thr), 4.22 (dd, 1H, 2J = 11.9 Hz, 3J = 5.6 Hz,
H-5′′), 4.06-4.03 (m, 1H, CH-β Thr), 2.60 (s, 3H, S-CH3), 2.11 (s, 3H,
3
1.24 (d, 3H, J = 6.6 Hz, CH3 Thr); 13C NMR: (DMSO-d6) δ: 170.99 (C=O
Thr), 165.02 (C-2), 154.45 (NH-CO-NH), 154.30 (C-4), 145.00 (C-8),
143.93 (C-6), 127.29 (C-5), 87.70 (C-1'), 85.73 (C-4'), 73.35 (C-2'), 70.31
(C-3'), 65.41 (CH-β Thr), 63.26 (CH-α Thr), 61.25 (C-5'), 20.18 (CH3 Thr),
14.12 (SCH3); ESI-HRMS calcd for [C16H20N6O7SNa]: 463.1012, found:
463.1010. (Spectra S33/S34 and Figure S8, Supporting Information).
3
CH3-CO), 2.07 (s, 3H, CH3-CO), 1.97 (s, 3H, CH3-CO), 1.18 (d, 3H, J =
7.0 Hz, CH3 Thr); 13C NMR (DMSO-d6) δ: 171.85 (C=O Thr), 169.96
This article is protected by copyright. All rights reserved.

10.1002/chem.201902411
Chemistry - A European Journal
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Compound 13b was prepared analogously as 13a starting from 12b and
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was obtained as a white solid (45 mg; 0.10 mmol; 92 % yield). Rf = 0.50
1
(n-BuOH/H2O, 85:15, v/v); H NMR: (DMSO-d6) δ: 8.73 (s, 1H, H-8), 8.68
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3
(bs, 1H, NH Thr), 6.00 (d, 1H, J = 5.6 Hz, H-1'), 5.57 (d, 1H, J = 6.3 Hz,
2'-OH), 5.33 (d, 1H, 3J = 4.2 Hz, 3'-OH), 5.23 (d, 1H, 3J = 4.9 Hz, -OH Thr),
5.00-4.99 (m,1H, 5'-OH), 4.70 (dd, 1H, 3J = 11.2 Hz, 3J = 5.6 Hz, H-2'), 4.42
(dd, 1H, 3J = 2.8 Hz, 3J = 1.4 Hz, CH-α Thr), 4.21 (dd, 1H, 3J = 9.1 Hz,
3J = 4.9 Hz, H-3'), 4.07-4.03 (m, 1H, CH-β Thr), 3.69 (dd, 1H, 3J = 7.7 Hz,
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170.18 (C=O Thr), 165.10 (C-2), 154.30 (NH-CO-NH), 154.27 (C-4), 145.18
(C-6), 143.78 (C-8), 127.43 (C-5), 87.72 (C-1'), 85.73 (C-4'), 73.35 (C-2'),
70.31 (C-3'), 66.36 (CH-β Thr), 62.83 (CH-α Thr), 61.24 (C-5'), 17.27 (CH3
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10.1002/chem.201902411
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FULL PAPER
Entry for the Table of Contents
Layout 2:
FULL PAPER
Katarzyna Debiec, Michal Matuszewski,
Karolina Podskoczyj, Grazyna
Leszczynska, Elzbieta Sochacka*
Page No. – Page No.
Chemical synthesis of
oligoribonucleotide (ASL of tRNALys T.
brucei) containing a recently
discovered cyclic form of
2-methylthio-N6-
Hydantoin modified ms2ct6A nucleoside was successfully introduced into the RNA
chain by post-synthetic ms2t6A→ms2ct6A cyclization at the oligonucleotide level.
The precursor ms2t6A-RNA was prepared using ms2t6A-phosphoramidite
monomeric unit obtained efficiently from adenosine or guanosine by the optimized
carbamate and, for the first time, the isocyanate chemistry.
threonylcarbamoyladenosine
(ms2ct6A)
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