Novel entry to the synthesis of (: S)- And (R)-5-methoxycarbonylhydroxymethyluridines-a diastereomeric pair of wobble tRNA nucleosides

Article abstract of DOI:10.1039/c9ra08548c

Two novel methods for the preparation of the virtually equimolar mixtures of (S)- and (R)-diastereomers of 5-methoxycarbonylhydroxymethyluridine (mchm⁵U) have been developed. The first method involved α-hydroxylation of a 5-malonate ester derivative of uridine (5) with SeO₂, followed by transformation to (S)- and (R)-5-carboxymethyluridines (cm⁵U, 8) and, finally, into the corresponding methyl esters. In the second approach, (S)- and (R)-mchm⁵-uridines were obtained starting from 5-formyluridine derivative (9) by hydrolysis of the imidate salt (11) prepared in the acid catalyzed reaction of 5-cyanohydrin-containing uridine (10b) with methyl alcohol. In both methods, the (S)- and (R) diastereomers of mchm⁵U were effectively separated by preparative C18 RP HPLC.

Full text of DOI:10.1039/c9ra08548c

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Novel entry to the synthesis of (S)- and (R)-5-
methoxycarbonylhydroxymethyluridines –
Cite this: RSC Adv., 2019, 9, 40507
a diastereomeric pair of wobble tRNA nucleosides†
Robert Borowski,
and Grazyna Leszczynska
Agnieszka Dziergowska,
Elzbieta Sochacka
*
Two novel methods for the preparation of the virtually equimolar mixtures of (S)- and (R)-diastereomers of
5-methoxycarbonylhydroxymethyluridine (mchm5U) have been developed. The first method involved a-
hydroxylation of a 5-malonate ester derivative of uridine (5) with SeO2, followed by transformation to (S)-
and (R)-5-carboxymethyluridines (cm5U, 8) and, finally, into the corresponding methyl esters. In the
second approach, (S)- and (R)-mchm5-uridines were obtained starting from 5-formyluridine derivative (9)
by hydrolysis of the imidate salt (11) prepared in the acid catalyzed reaction of 5-cyanohydrin-containing
uridine (10b) with methyl alcohol. In both methods, the (S)- and (R) diastereomers of mchm5U were
effectively separated by preparative C18 RP HPLC.
Received 18th October 2019
Accepted 28th November 2019
DOI: 10.1039/c9ra08548c
rsc.li/rsc-advances
sensitivity to DNA-damaging agents.11 This observation indi-
cates possible connection between a regulatory mechanism in
Introduction
DNA/RNA damage response pathways and modulation of tRNA
modication. Interestingly, a negative correlation was discov-
ered between the level of ALKBH8 and urothelial cancer
progression.12 Additionally, some recessive truncating muta-
tions in human ALKBH8 gene were recently shown to cause
intellectual disability associated with the absence of (S)-
mchm5U, (R)-mchm5U, m5U, mcm5Um or mcm5s2U units in
total tRNA.13
Undoubtedly, efficient and reliable methods of synthesis of
stereochemically dened nucleosides 1 and 2 would facilitate
research on the biological activities of ALKBH proteins, and on
the unknown path of introduction of (R)-mchm5U units to the
mammalian tRNAAUrCgG. The methods reported to date are based
on a regioselective C5-lithiation of the uracil residue with n-
BuLi and a subsequent reaction of the heterobase C5-carbanion
with ethyl or butyl glyoxylate (H(O)CCH2COOR, R ¼ Bu, Et).6,7,14
Kawakami and co-workers utilized the coupling of mchm5-Ura
with a ribose unit using the Vorbruggen's method.7 Next two
5-Substituted uridines constitute a class of biologically impor-
tant modied nucleosides located predominantly at the wobble
position 34 (the rst anticodon letter) of transfer RNAs from all
domains of life.1 Wobble uridines are essential for efficient and
accurate decoding of genetic information.2,3 Their absence oen
leads to translation defects and severe diseases.3,4
Wobble (S)- and (R)-5-methoxycarbonylhydroxymethylur-
idines ((S)-mchm5U, 1, (R)-mchm5U, 2, Fig. 1), represent
a unique example of a diastereomeric pair of modied tRNA
nucleosides. In mammals, the (S)-isomer 1 was identied in
Gly
UCC
Arg
5,6
tRNAs
while (R)-mchm5U (2) in tRNAs
.
UCG
Notably, (S)-
mchm5U (1) was also detected in tRNAsGlyUCC from insects (e.g.
B. mori), worms (e.g. C. elegans) and plants (e.g. A. thaliana).5,7–9
tRNAs bearing either of these diastereomeric nucleosides are
derived from the corresponding tRNAs containing 5-methox-
ycarbonylmethyluridine (mcm5U, 3, Fig. 1).5,6 In mammalian
tRNAs, the cm5U34 / mcm5U34 methyl transfer reaction, as
well as subsequent stereoselective oxidation (C–H / C–OH
conversion) to (S)-mchm5U34, are catalyzed by an ALKBH8
enzyme,5,6 the member of an AlkB protein family,10 which are
mainly involved in DNA/RNA repair processes.5,6 Human cells
deprived of ALKBH8 were found to have reduced the endoge-
nous level of mcm5U tRNA wobble modication and increased
Institute of Organic Chemistry, Lodz University of Technology, Zeromskiego 116,
90-924 Lodz, Poland. E-mail: grazyna.leszczynska@p.lodz.pl; Fax: +4842 636 55 30;
Tel: +4842 631 31 50
† Electronic supplementary information (ESI) available: 1H and 13C spectra of
compounds 1, 2, 5–8, and 10a–b; RP-HPLC chromatogram of 1 and 2 mixture,
CD spectra of 1 and 2. See DOI: 10.1039/c9ra08548c
Fig. 1 Chemical structures of (S)-5-methoxycarbonylhydroxymethylur-
idine ((S)-mchm5U, 1), (R)-5-methoxycarbonylhydroxymethyluridine ((R)-
mchm5U, 2) and 5-methoxycarbonylmethyluridine (mcm5U, 3).
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procedures involved C5-lithiation of uridine14 or 5-bromour- (Scheme 1B).6 Noteworthy, 5 could be used in preparation of all
idine6 with the regioselectivity controlled by TBDMS protection nucleosides 1–3 employing the same sequence of reactions:
of the nucleoside sugar moiety. The C5-lithiated species were deprotection, decarboxylation and methyl esterication.
then treated with butyl or ethyl glyoxylate to give a mixture of
The “malonate” derivative 5 was obtained in a DBU promoted
fully protected diastereomers, which were separated by chro- reaction of 20,30,50-O-triacetyl-N3-benzoyl-5-bromouridine (4) and
matographic methods. Subsequent methanolysis of each diethyl malonate (ESI, Fig. S1 and S2†).6,15 The C-5,1-oxidation
TBDMS-protected isomer provided pure nucleosides 1 and 2. upon treatment with SeO2 was optimized in terms of molar
However, because of restrictive conditions of the lithiation and excess (1.5–4 equiv.), temperature (25–100 ꢀC), solvent (1,4-
unavoidable partial polymerization of glyoxylate, the afore- dioxane, t-BuOH, t-BuOH/1,4-dioxane, CH2Cl2 in the presence of
mentioned protocols are rather poorly reproducible.
t-BuOOH as co-oxidant), and reaction time (5–48 h).16 The best
In this work, we present two novel methods for preparation yield of the hydroxyl derivative 6 was achieved using 4 equivalents
of 1 and 2, where instead of the formation of the uridine C5- of SeO2 in boiling 1,4-dioxane for 18 h. It was isolated by chro-
carbanion, the nal modication was introduced by the trans- matography on a silica gel column in 68% yield and its structure
formation of nucleoside substrates containing a methyl-derived was conrmed by NMR and MS data (ESI, Fig. S3 and S4†).
substituent at C5-uracil position (so-called C-5,1-functionalized
Unfortunately, our subsequent attempts at deprotection (alka-
uridines), namely 5-(diethyl malonate-yl) derivative of protected line hydrolysis of acetyl, benzoyl, and ethyl ester groups) and
uridine 5 (Scheme 1) or 5-formyluridine derivative 9 (Scheme 2). simultaneous decarboxylation in 6 using 2.2 M MeONa–MeOH at
Notably, 5-formyluridine 9 was converted to 1–2 via a 5-cyano- 50 ꢀC (a one-pot procedure reported by Fu for a 5 / 3 conversion,
hydrin derivative, the synthesis and useful reactivity of which is Scheme 1B)6 led to a complex mixture of products. Thus, to
reported for the rst time.
perform only the deprotection, we treated 6 with 2.2 M NaOMe–
MeOH at room temperature.15 Aer 20 h, virtually quantitative
formation of a highly polar compound was noted, which was
deprived of acetyl and benzoyl groups, but contained one
methoxycarbonyl substituent (ESI, Fig. S5 and S6†). Because in the
relevant 1H and 13C NMR spectra certain resonance lines were
doubled (ESI, Fig. S5 and S6†) we assumed the formation of dia-
stereomeric species. Indeed, NMR and MS data allowed to identify
a cyclic intermediate 7 bearing a new stereogenic centre (marked
with an asterisk), which presumably was formed in independent
reactions of –COOEt / –COOMe conversion and g-lactonization,
the latter with the participation of the O4-atom. To open the
lactone and to perform subsequent decarboxylation, crude 7 was
treated with 50% aqueous triuoroacetic acid (TFA) at 60 ꢀC.15
Aer 15 h, TLC analysis showed the presence of a new product,
which was even more polar than the parent 7. It was isolated by
chromatography on an open column (C8-RP) in 50% yield (calcu-
lated over compound 6) and identied as a ca. 1 : 1 mixture of (S)-
Results and discussion
Our rst approach to the preparation of 1 and 2 was based on the
assumption that the pivotal OH group in mchm5U can be
introduced by selective SeO2-mediated oxidation of C-5,1 carbon
in 20,30,50-tri-O-acetyl-N3-benzoyl-5-(bis(ethoxycarbonyl)methyl)-
uridine (5) (“malonate” derivative, Scheme 1A). We appreciated
the accessibility of the “malonate” derivative (5), which was
a substrate in synthesis of 5-methoxycarbonylmethyluridine (3)
and (R)-5-carboxyhydroxymethyluridine
8 (ESI, Fig. S7–S9†).
Treatment of the mixture of (S)- and (R)-acids 8 with anhydrous
1 M HCl–MeOH at room temperature for 2 h furnished the mixture
of (S)- and (R)-mchm5U diastereomers (1–2) (ESI, Fig. S10–S13†),
which was resolved onto stereochemically pure species by RP
HPLC in 43% and 50% yield, respectively (a preparative C18
column was eluted with water as shown in ESI, Fig. S17A;†
conditions for separation of diastereomers were established based
on the analytical HPLC, see ESI, Fig. S17B†).
Our second approach to the preparation of 1 and 2 was based
on the prediction that 5-formyluridine 9 can be effectively
transformed to the appropriate 5-cyanohydrin derivative and
then to the imidate salt by a Pinner reaction (Scheme 2).
The recent development of 5-formylpyrimidine nucleosides
Scheme 1 (A) Synthesis of (S)- and (R)-5-methoxycarbonylhydrox- as epigenetic modications signicantly improved the synthetic
availability of these nucleosides.17 In the present research, we
used our earlier reported method, based on selective oxidation
of the appropriately protected 5-hydroxymethyluridine with
activated MnO2,18 to prepare a 5-formyluridine derivative 9
(Scheme 2). Initial experiments aimed at the conversion of the
ymethyluridine (1 and 2) with 5-malonylated uridine 5 as a key inter-
mediate. Reagents and conditions: i/diethyl malonate, DBU, THF, rt,
15 h; ii/SeO2, dioxane, reflux, 18 h; iii/2.2 M MeONa/MeOH, MeOH, rt,
20 h; iv/TFA : H2O (1 : 1, v/v), 60 ꢀC, 15 h; v/1 M HCl/MeOH, rt, 2 h; (B)
Synthetic route to 5-methoxycarbonylmethyluridine (3) according to
Fu et al.6 i/MeONa/MeOH, 50 ꢀC, 16 h.
40508 | RSC Adv., 2019, 9, 40507–40512
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The stereochemical assignments of (S)- and (R)-mchm5U (1
and 2, respectively) were conrmed by comparison of their CD
spectra (ESI, Fig. S18†) with those reported earlier by Nawrot
and Fu.6,14
Conclusions
Two new, more reliable methods of preparation of the mixture
of (S)-mchm5U (1) and (R)-mchm5U (2), based on the use of
C5,1-functionalized uridines (the “malonate” 5 and formyl 9
derivatives) as the substrates, with subsequent chromato-
graphic separation of the diastereomers, have been reported.
These methods signicantly improved the availability of these
synthetically demanding nucleosides and will facilitate research
on their presence and functions in native tRNAs and on the
unique activities of the ALKB-like proteins involved in modu-
lation of tRNA functions.
Scheme 2 The synthesis of (S)- and (R)-5-methoxycarbonylhydroxy-
methyluridine (1 and 2) starting with 5-formyluridine 9. Reagents and
conditions: i/TBDMSCN, NEt3, CH3CN, rt, 2 h; ii/4 M HCl/MeOH, 5 ꢀC,
2 h; iii/H2O, 5 ꢀC, 2 h.
Experimental
General methods
Thin layer chromatography was done on silica gel coated plates
(60F254, Merck), and Merck silica gel 60 (mesh 230–400, Merck)
or Fluka silica gel 100 C8-RP were used for column chroma-
tography. HPLC was performed with a Waters chromatograph
equipped with a 996 spectral diode array detector and
a preparative Ascentis® column (C18, 25 cm ꢁ 21.2 mm, 10 mm,
SUPELCO). Separation was run at rt using water as an eluent.
NMR spectra were recorded on 250 MHz or 700 MHz instru-
ments (176 MHz for 13C NMR). Chemical shis (d) are reported
5-formyl group into a cyanohydrin function (C5–CH(CN)OH)
using KCN/acetic acid19 failed because several attempts at
isolation of the product ended with predominant recovery of the
substrate. This reversibility was debarred by the use of TBDMS-
cyanide20 (up to 2-fold molar excess), so the hydroxyl group of
the cyanohydrin function was instantly protected with a TBDMS
group (–C5–CH(CN)OTBDMS). However, aer 15 minutes, two
products were observed regardless of the reagent ratio. The
relevant NMR spectra revealed that the desired O-silylated
cyanohydrin adduct (10a, ESI, Fig. S14†) was accompanied by its
derivative 10b bearing also 50-O-TBDMS group (ESI, Fig. S15 and
S16†). Our attempts to increase the selectivity of the cyanohy-
drin protection were unsuccessful. Therefore, using 3-fold
molar excess of TBDMS-CN the bis-silylated compound 10b was
obtained (in 80% yield) and used for further transformations.
Hydrogen chloride catalyzed addition of methanol to the CN
group in 10b (the Pinner reaction)21,22 led to an imidate hydro-
chloride salt 11 (Scheme 2), from which residual HCl was
carefully removed to have the conditions safe for the methyl
ester group present in the nal nucleoside. Subsequent simple
hydrolysis (the treatment with water at 5 ꢀC for 2 h)21,22 fur-
nished a mixture of isomers of mchm5U (1–2). Their identity
was conrmed by HPLC comparison with the reference
samples. The isomeric products were separated by RP HPLC (a
preparative C18 column, water as an eluent) and each diaste-
reomer was obtained in ca. 25% yield (the 50% yield obtained
for both diastereomers refers to 10b).
1
in ppm relative to TMS (an internal standard) for H and 13C.
The signal multiplicities are described as s (singlet), d (doublet),
dd (doublet of doublets), t (triplet), q (quartet), m (multiplet),
and bs (broad singlet). High-resolution mass spectrometry
(HRMS) spectra were recorded using a Synapt G2Si mass spec-
trometer (Waters) equipped with an ESI source and a quadru-
pole-time-of-ight mass analyzer. The measurements were
performed in a negative ion mode and the results were pro-
cessed using the MassLynx 4.1 soware (Waters). CD spectra
were recorded on Spectrometer CD J-1000 JASCO in a 1 ml
quartz cell. UV measurements were made using a Specord®50
PLUS spectrophotometer.
20,30,50-tri-O-acetyl-N3-benzoyluridine-5-malonic acid diethyl
ester (5)
To the solution of 20,30,50-tri-O-acetyl-N3-benzoyl-5-bromouridine
(4)15 (1.74 g, 3.15 mmol, 1 equiv.) in anhydrous THF (18 ml),
diethyl malonate (535 ml, 3.5 mmol, 1.1 equiv.) and DBU (670 ml,
4.5 mmol, 1.4 equiv.) were added. The reaction mixture was stirred
at room temperature for 15 h and then neutralized with AcOH. The
solvent was evaporated under reduced pressure. The residue was
dissolved in ethyl acetate (20 ml) and washed with water and brine.
The organic layer was separated, dried over MgSO4 and concen-
trated in vacuo. The solid residue was applied on a silica gel
column, further eluted with CHCl3 to afford compound 5 in 78%
yield (1.55 g). TLC Rf ¼ 0.29 (CH2Cl2/acetone 98 : 2, v/v). NMR (d
As expected, the reagent used in the Pinner reaction (4 M
HCl/MeOH,
2
h) concomitantly removed the 20,30-iso-
propylidene and TBDMS protecting groups. The use of 2 M HCl/
MeOH led to longer reaction time (4 h) while 1 M HCl/MeOH
resulted in the recovery of 5-formyluridine, probably because
of the preference for TBDMS-cyanohydrin deprotection over the
imidate salt formation.
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[ppm], CDCl3): 1H (700 MHz) 7.98 (s, 1H), 7.94–7.95 (m, 2H), 7.64–
7.66 (m, 1H), 7.49–7.51 (m, 2H), 6.18 (d, 1H, J ¼ 5.75 Hz), 5.39–5.43
(m, 2H), 4.87 (s, 1H), 4.35–4.38 (m, 3H), 4.17–4.26 (m, 4H), 2.16 (s,
3H), 2.12 (s, 3H), 2.04 (s, 3H), 1.28 (t, 3H, J ¼ 7.00 Hz), 1.27 (t, 3H, J
¼ 7.50 Hz). 13C (176 MHz) 170.26, 169.34, 169.23, 167.43, 166.92,
166.87, 160.74, 148.40, 139.13, 134.84, 130.80, 130.27, 128.80,
107.51, 87.07, 80.28, 72.60, 70.42, 62.97, 62.05, 46.76, 20.25, 20.15,
20.00, 13.58; HRMS calcd for C29H31N2O14 [M ꢂ H]ꢂ 631.1775,
found 631.1769 (ESI, Fig. S1 and S2†).
(S)- and (R)-5-carboxyhydroxymethyluridine (8)
The remaining amount of crude compound 7 (vide supra) was
dissolved in 50% aqueous TFA (3.4 ml). The solution was stirred
ꢀ
for 15 h at 60 C and concentrated in vacuo. The residue was
puried on a column of silanized silica gel (100 C8-RP) eluted
with water. Compound 8 was obtained in 50% yield (30 mg, the
yield refers to the starting compound 6). TLC Rf ¼ 0.63 (iPrOH/
1
H2O, 7 : 3, v/v, for both diastereoisomers); NMR (d [ppm]): H
(700 MHz, D2O) 8.04 (s, 0.5H), 8.03 (s, 0.5H), 5.85 (d, 0.5H, J ¼
4.2 Hz), 5.86 (d, 0.5H, J ¼ 4.2 Hz), 5.03 (s, 0.5H), 5.04 (s, 0.5H),
4.28–4.30 (m, 1H), 4.17–4.19 (m, 1H), 4.07–4.08 (m, 1H), 3.88
(dd, 1H, J ¼ 2.8 Hz, J ¼ 12.6 Hz), 3.74–3.77 (m, 1H), H (700
20,30,50-tri-O-acetyl-N3-benzoyluridine-5-a-hydroxymalonic acid
diethyl ester (6)
1
MHz, DMSO-d6) 11.44 (s, 0.5H), 11.43 (s, 0.5H), 7.90 (s, 0.5H),
7.88 (s, 0.5H), 5.82 (s, 0.5H), 5.81 (s, 0.5H), 5.58 (bs, 1H), 5.38–
5.41 (m, 1H), 5.11 (bs, 1H), 5.04 (bs, 1H), 4.75 (s, 0.5H), 4.74 (s,
0.5H), 4.02–4.06 (m, 1H), 3.94–3.98 (m, 1H), 3.85–3.88 (m, 1H),
3.53–3.65 (m, 2H). 13C (176 MHz, D2O) 174.50, 163.40 (163.35),
150.90, 140.69, 112.13, 89.26 (89.21), 83.70, 73.52, 68.70 (68.69),
66.45 (66.38), 60.01 (59.97); HRMS calcd for C11H13N2O9 [M ꢂ
H]ꢂ 317.0621, found 317.0623 (ESI, Fig. S7–S9†).
To a stirred solution of 20,30,50-tri-O-acetyl-N3-benzoyluridine-5-
malonic acid diethyl ester (5) (460 mg, 0.73 mmol, 1 equiv.) in
1,4-dioxane (6.4 ml), solid SeO2 (324 mg, 2.92 mmol, 4 equiv.)
was added and the reaction mixture was reuxed for 18 h. The
mixture was cooled down to room temperature and ltered. The
ltrate was concentrated under reduced pressure, the residue
was dissolved in ethyl acetate (10 ml) and washed with saturated
NaHCO3, water and brine. The organic layer was dried over
MgSO4 and concentrated. The product 6 was isolated by silica
gel column chromatography (elution with 1–20% AcOEt in
CH2Cl2) in 68% yield (322 mg). TLC Rf ¼ 0.80 (AcOEt : CH2Cl2
15 : 85, v/v). NMR (d [ppm], DMSO-d6): 1H (250 MHz) 7.94–8.00
(m, 3H), 7.80–7.96 (m, 1H), 7.61–7.67 (m, 2H), 7.38 (s, 1H), 6.00
(d, 1H, J ¼ 4.5 Hz), 5.53 (dd, 1H, J ¼ 4.75 Hz, J ¼ 6.25 Hz), 5.34–
5.39 (m, 1H), 4.17–4.37 (m, 3H), 4.12 (q, 2H, J ¼ 7.25 Hz), 2.06 (s,
3H), 2.05 (s, 3H), 2.02 (s, 3H), 1.13 (t, 3H, J ¼ 7.25 Hz). 13C (176
MHz) 170.17, 169.54, 169.51, 169.39, 168.58, 168.36, 159.88,
148.43, 139.58, 139.92, 130.70, 130.38, 129.61, 113.38, 89.32,
79.55, 76.49, 72.68, 69.56, 62.64, 61.69, 20.49, 20.37, 20.34,
13.77; HRMS calcd for C29H31N2O15 [M ꢂ H]ꢂ 647.1724, found
647.1724 (ESI, Fig. S3 and S4†).
Conversion of 8 into (S)-5-methoxycarbonylhydroxymethyluridine
(1) and (R)-5-methoxycarbonylhydroxymethyluridine (2)
5-Carboxyhydroxymethyluridine (8) (27 mg, 0.085 mmol) was
dissolved in 1 M HCl/MeOH (1.6 ml). The solution was stirred at
room temperature for 2 h and concentrated in vacuo. The
residue was co-evaporated three times with anhydrous toluene
and, nally, with methanol. A sample was applied on a prepar-
ative C18 column (SUPELCO; Ascentis®, 25 cm/21.2 mm; 10
mm; ow 6 ml minꢂ1) further eluted with water. Aer HPLC
separation, 12 mg (43%) of (S)-mchm5U (1) and 14 mg (50%) of
(R)-mchm5U (2) were obtained (tR ¼ 7.9 min and 9.4 min,
respectively). TLC: Rf ¼ 0.45 (n-BuOH/H2O, 85 : 15, v/v, for both
1
diastereoisomers). Spectral data for 1: NMR (d [ppm], D2O): H
(700 MHz) 8.13 (s, 1H), 5.96 (d, 1H, J ¼ 4.2 Hz), 5.15 (s, 1H), 4.39
(dd, 1H, J ¼ 4.2 Hz, J ¼ 5.6 Hz), 4.28 (t, 1H, J ¼ 5.6 Hz), 4.18–4.20
(m, 1H), 3.90 (dd, 1H, J ¼ 2.8 Hz, J ¼ 12.6 Hz), 3.86 (dd, 1H, J ¼
4.2 Hz, J ¼ 12.6 Hz), 3.82 (s, 3H). 13C NMR (176 MHz) 173.69,
163.81, 151.36, 141.14, 112.45, 89.64, 84.09, 73.89, 69.09, 67.08,
60.40, 53.14; HRMS calcd for C12H15N2O9 [M ꢂ H]+ 331.0778,
found 331.0783. Spectral data for 2: NMR (d [ppm], D2O): 1H
(700 MHz) 8.14 (s, 1H), 5.96 (d, 1H, J ¼ 4.2 Hz), 5.16 (s, 1H), 4.40
(dd, 1H, J ¼ 4.2 Hz, J ¼ 5.6 Hz), 4.29 (t, 1H, J ¼ 5.6 Hz), 4.18–4.19
(m, 1H), 3.99 (dd, 1H, J ¼ 2.8 Hz, J ¼ 12.6 Hz), 3.86 (dd, 1H, J ¼
3.5 Hz, J ¼ 12.6 Hz), 3.82 (s, 3H). 13C (176 MHz) 173.68, 163.82,
151.39, 141.23, 112.34, 89.69, 84.02, 73.78, 69.02, 67.05, 60.31,
53.14; HRMS calcd for C12H15N2O9 [M ꢂ H]ꢂ 331.0778, found
Cyclic intermediate – the lactone 7
To a solution of 6 (123 mg, 0.19 mmol, 1 equiv.) in anhydrous
MeOH (2.8 ml), 2.2 M solution of MeONa in MeOH (370 ml) was
added and the mixture was stirred at room temperature for 20 h.
Then the mixture was diluted with MeOH (3 ml) and neutralized
by DOWEX 50W (H+ form) resin. The resin was ltered off and
the ltrate was concentrated. For spectral analyses small amount
(ca. 15 mg) of the solid residue was applied on a column of
silanized silica gel (100 C8-RP). The column was eluted with water
to afford analytically pure 7. Because of the presence of diaste-
reomers, some 13C NMR resonances were doubled (the secondary
shis in the 13C NMR spectrum are given in parentheses). TLC Rf
¼ 0.82 (iPrOH/H2O, 7 : 2, v/v). NMR (d [ppm], D2O): 1H (250 MHz)
7.96 (s, 0.5H), 7.95 (s, 0.5H), 5.92 (d, 0.5H, J ¼ 1.5 Hz), 5.91 (d,
0.5H, J ¼ 1.75 Hz), 4.27–4.31 (m, 1H), 4.09–4.20 (m, 2H), 3.87–
3.92 (m, 1H), 3.79 (s, 3H), 3.72–3.77 (m, 1H). 13C (176 MHz)
171.76 (171.66), 170.89 (170.85), 163.33, 150.79 (150.77), 139.49
331.0784; UV (H2O) lmax ¼ 266 (3266 ¼ 9553 l molꢂ1 cmꢂ1, 3260
8592 l molꢂ1 cmꢂ1) (ESI, Fig. S10–S13†).
¼
(S)- and (R)-20,30-O-isopropylidene-50-O-tert-butyldimethylsilyl-
5-(O-tert-butyldimethylsilyl)cyanohydroxymethyluridine (10b)
(139.46), 113.35 (133.23), 89.30 (89.24), 83.76 (83.74), 77.96 To a solution of 20,30-O-isopropylidene-5-formyluridine (9)18
(77.94), 73.74 (73.65), 69.08 (69.04), 60.40 (60.39), 53.28 (53.27) (1.55 g, 4.98 mmol, 1 equiv.) in anhydrous acetonitrile (62 ml),
(ESI, Fig. S5 and S6†).
TBDMS-CN (2.09 g, 14.94 mmol, 3 equiv) and triethylamine
40510 | RSC Adv., 2019, 9, 40507–40512
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(2.06 ml, 14.94 mmol, 3 equiv.) were added. The mixture was
stirred at room temperature for 2 h and concentrated under
reduced pressure. The crude product was puried by silica gel
column chromatography using a 0–5% gradient of methanol in
chloroform as an eluent. Compound 10b was obtained in 80%
yield (2.26 g). Because of the presence of diastereomers some
13C NMR resonances were doubled (the secondary shis in the
13C NMR spectrum are given in parentheses). Rf ¼ 0.29 (CH2Cl2/
acetone 98 : 2, v/v), 0.53 (hexane/ethyl acetate 3 : 1 v/v). NMR (d
[ppm], CDCl3): 1H (700 MHz) 0.06 (s, 1.5H), 0.08 (s, 1.5H), 0.08
(s, 3H), 0.18 (s, 1.5H), 0.19 (s, 1.5H), 0.25 (s, 1.5H), 0.27 (s, 1.5H),
0.86 (s, 4.5H), 0.88 (s, 4.5H), 0.93 (s, 9H), 1.36 (s, 1.5H), 1.37 (s,
1.5H), 1.58 (s, 3H), 4.33–4.35 (m, 0.5H), 4.44–4.45 (m, 0.5H),
4.72–4.73 (m, 0.5H), 4.88–4.90 (m, 1H), 5.48 (s, 0.5H), 5.50 (s,
0.5H), 5.69 (d, 0.5H, J ¼ 2.37 Hz), 5.71 (d, 0.5H, J ¼ 2.46 Hz), 7.74
(s, 0.5H), 7.82 (s, 0.5H), 8.90 (s, 0.4H), 8.86 (s; 0.4H); 13C (176
MHz) ꢂ4.60 (ꢂ4.49), ꢂ4.35 (ꢂ4.23), 19.11 (19.16), 19.28 (19.36),
26.15 (25.24), 26.51 (26.56), 26.91 (26.85), 29.08 (29.16), 58.39
(58.47), 64.76 (64.66), 82.51 (82.26), 85.99 (86.70), 88.79 (89.06),
96.53 (96.09), 110.82 (111.55), 114.76 (115.12), 118.61 (118.66),
140.64 (141.43), 150.38 (150.43), 161.87 (161.90). HRMS calcd
for C26H45N3O7Si [M ꢂ H]ꢂ 567.2796, found 567. 2722 (ESI,
Fig. S15 and S16†).
Notes and references
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Conversion of 10b into (S)-5-methoxycarbonylhydroxymethyl-
uridine (1) and (R)-5-methoxycarbonylhydroxymethyluridine (2)
Compound 10b (80 mg, 0.14 mmol) was dissolved in cold (an ice
bath) 4 M HCl/MeOH (8 ml, prepared by dropwise addition of
AcCl to cold anhydrous MeOH). The reaction vessel was moved
to a refrigerator (set at 5 ꢀC) and the mixture was stirred for 2 h
(TLC control, isopropanol/water 7 : 3, v/v, Rf ¼ 0.88). The vola-
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Conflicts of interest
There are no conicts to declare.
Acknowledgements
This work was supported by the statutory funds of Lodz
University of Technology. The authors thank Dr Piotr Guga for
critical reading of the manuscript.
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