Transformation of a wobble 2-thiouridine to 2-selenouridine via S-geranyl-2-thiouridine as a possible cellular pathway

Article abstract of DOI:10.1016/j.bioorg.2014.05.012

The newly discovered S-geranylated 2-thiouridines (geS2U) (Dumelin et al., 2012) and 2-selenouridines (Se2U) were recently shown to be synthesized by a single enzyme (selenouridine synthase, SelU) through two distinct pathways using the same 2-thiouridine substrate (S2U); however, no clear catalytic mechanism was proposed. We suggest that S-geranyl-2-thiouridine is an intermediate of the SelU-catalyzed conversion of S2U to Se2U. The successful chemical transformation of S2U → geS2U → Se2U is demonstrated here as an initial approximation of the intracellular pathway. The structure of Se2U was confirmed by spectroscopic methods, which included, for the first time, ⁷⁷Se NMR data (δ 354 ppm).2014 Elsevier Inc. All rights reserved.

Full text of DOI:10.1016/j.bioorg.2014.05.012

Accepted Manuscript
Transformation of a wobble 2-thiouridine to 2-selenouridine via S-geranyl-2-
thiouridine as a possible cellular pathway
Paulina Bartos, Anna Maciaszek, Anna Rosinska, Elzbieta Sochacka, Barbara
Nawrot
PII:
S0045-2068(14)00045-5
DOI:
http://dx.doi.org/10.1016/j.bioorg.2014.05.012
YBIOO 1730
Reference:
Bioorganic Chemistry
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Received Date:
5 March 2014
Please cite this article as: P. Bartos, A. Maciaszek, A. Rosinska, E. Sochacka, B. Nawrot, Transformation of a wobble
2-thiouridine to 2-selenouridine via S-geranyl-2-thiouridine as a possible cellular pathway, Bioorganic Chemistry
(2014), doi: http://dx.doi.org/10.1016/j.bioorg.2014.05.012
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Transformation of a wobble 2-thiouridine to 2-selenouridine via S-geranyl-2-
thiouridine as a possible cellular pathway
Paulina Bartos,a Anna Maciaszek,b Anna Rosinska,b Elzbieta Sochacka,a and Barbara Nawrotb*
aInstitute of Organic Chemistry, Faculty of Chemistry, Technical University of Lodz, Zeromskiego 116,
90-924 Lodz, bDepartment of Bioorganic Chemistry, Centre of Molecular and Macromolecular Studies,
Polish Academy of Sciences, Sienkiewicza 112, 90-363 Lodz, Poland;
* Corresponding author. Address: Barbara Nawrot, Centre of Molecular and Macromolecular Studies,
Polish Academy of Sciences, Sienkiewicza 112, 90-363 Lodz, Poland. Phone: +48-42-6816970; Fax:
+48-42-6815483
E-mail address: bnawrot@cbmm.lodz.pl
Paulina Bartos p.bartos@o2.pl
Dr Anna Maciaszek annamck@cbmm.lodz.pl
Anna Rosinska arosinsk@cbmm.lodz.pl
Prof. Elzbieta Sochacka elzbieta.sochacka@p.lodz.pl
Abstract
The newly discovered S-geranylated 2-thiouridines (geS2U) (Dumelin et al, Nat. Chem. Biol. 2012)
and 2-selenouridines (Se2U) were recently shown to be synthesized by a single enzyme
(selenouridine synthase, SelU) through two distinct pathways using the same 2-thiouridine substrate
(S2U); however, no clear catalytic mechanism was proposed. We suggest that S-geranyl-2-thiouridine
is an intermediate of the SelU-catalyzed conversion of S2U to Se2U. The successful chemical
transformation of S2U→geS2U→Se2U is demonstrated here as an initial approximation of the
intracellular pathway. The structure of Se2U was confirmed by spectroscopic methods, which
included, for the first time, 77Se NMR data (δ354 ppm).
Keywords: 2-thiouridine, 2-selenouridine, S-geranyl-2-thiouridine, selenouridine synthase, SelU, S-
alkylation, selenium chemical shift, 77Se NMR
1. Introduction
Transfer RNAs (tRNAs) are unique among natural RNAs because of their substantial content of
modified nucleosides, including 5-substituted uridines (x5U, 1) and their analogs containing either a
sulfur (2-thiouridines, x5S2U, 2) or selenium atom (2-selenouridines, x5Se2U, 3) in the nucleobase C2
1

position. These hypermodified nucleosides are located in the wobble position of the anticodon
(position 34) of tRNAs specific for glutamine, glutamic acid and lysine [1,2]. Their structural features
play a fundamental role in the tuning of codon reading and the efficiency of the translation process
[3]. Ten type 2 2-thiouridines and five type 3 2-selenouridines are known; the substituents at C5 (X)
are shown in Fig. 1.
O
O
O
O
X
X
X
NH
X
NH
NH
N
O
S
N
N
O
O
Se
N
N
S
HO
HO
O
O
HO
HO
HO
OH
HO
OH
HO
OH
HO
OH
x5U (1)
x5S2U (2)
x5Se2U (3)
x5geS2U (4)
Substituent x
—H
—CH3
—CH2NH2
—CH2NHCH3
—CH2NHCH2COOH
—CH2NHCH2CH2SO3
—CH2NHCH2CH=C(CH3)2
—CH2COOH
Notation for x
2
a
b
c
d
e
f
3
a
-
c
d
e
-
4
a
-
m
nm
mnm
cmnm
τm
-
d
e
-
inm
g-
-
-
-
-
cm
mcm
f
h
i
j
-
-
j
—CH2COOCH3
—CHO
Figure 1. Structures of the known 5-substituted uridines (x5U, 1), 2-thiouridines (x5S2U, 2), 2-
selenouridines (x5Se2U, 3) and S-geranyl-2-thiouridines (x5geS2U, 4).
Two hydrophobic 5-substituted-2-thiouridines in which the sulfur atom is functionalized with a
geranyl residue (mnm5geS2U, 4d and cmnm5geS2U, 4e) were recently identified in several bacterial
strains at position 34 of tRNAs specific for lysine, glutamine and glutamic acid [4]. tRNA geranylation
is induced by cellular stress and occurs in less than 7% of the cell population. The geranylation of the
wobble S2U nucleoside affects the codon preference and frame shifting during translation. Although
the exact mechanism of this effect is unknown, the superior recognition of the G-ending over the A-
ending codons by geranylated tRNALys is observed.
2

Selenouridine synthase SelU is the enzyme responsible for selenoation of 2-thiouridine in E. coli [5],
and Liu and co-workers have demonstrated that this enzyme is also involved in the geranylation of 2-
thiouridine [4]. These two chemically disparate processes employ the same 2-thiouridine substrate.
For selenoation, the sulfur atom in 2-thiouracil must be converted into a leaving group and further
substituted by a selenium nucleophile originating from selenophosphate. By contrast, for
geranylation, the same sulfur atom acts as a nucleophile and accepts the geranyl substituent from
geranyl diphosphate. It is unclear how these two mechanistically different reactions are mediated by
the same catalyst. Moreover, the potential relationship between the two distinct products, Se2U and
geS2U, formed from the same precursor is also of interest.
The literature suggests that the chemical transformation of S2U to various C2-substituted uridines
might occur through the respective S-alkylated derivatives. For example, S-alkylation of 2-thiouridine
yielded a series of 2-alkyl(aryl)aminouridines [6]. In addition, S-methylated 2’-deoxy-2-thiouridine
was successfully transformed (via a hydrazino derivative) into a 2-azidonucleoside [7]. Huang et al.
demonstrated that S-methylated 2-thiouridine is efficiently transformed to 2-selenouridine upon
nucleophilic attack of the hydroselenide ion (HSe-) on the partially electropositive C2 center [8].
Accordingly, we hypothesized that the newly discovered S-geranyl-2-thiouridine is an intermediate
formed during the transformation of 2-thiouridine (embedded in a tRNA chain) to 2-selenouridine.
We therefore attempted the chemical transformation S2U→geS2U→Se2U as an initial approximation
of the intracellular mechanism.
2. Results
The S-geranylated 5’-O-DMT-2-thiouridine substrate (6) was obtained from 2-thiouridine (2a) either
through its geranylated form 4a [4] via subsequent dimethoxytritylation at the 5’-OH function or via
5’-OH protection with a DMT group, yielding compound 5, and subsequent geranylation of the sulfur
1
residue (Scheme 1). The structure of S-geranyl-2-thiouridine 2a was confirmed by H and 13C NMR
and FAB-MS spectral analysis (for numbering, see Supplementary Material Fig. S2; for spectral
analysis, see Fig. S3 - S5). The 5’-DMT protection of 4a was accomplished using standard protocols to
give pure 6. A similar approach was used for geranylation prior to 5’-OH protection (2a →5 →6) (see
1
the Supplementary Material for preparative procedures and H NMR spectral data of 5, Fig. S6, S7
for). The efficiencies of the first and second synthetic approaches were similar and produced pure
1
derivative 6 in 66 and 46% yield, respectively. The structure of 6 was confirmed by H and 13C NMR
and FAB mass spectrometry (Fig. S8 - S13). The DMT group improved the solubility of 4a in organic
solvents, thus facilitating the subsequent selenoation.
3

O
N
O
N
NH
N
geranyl bromide
MeOH
HO
S
HO
S
O
O
HO
OH
2a
HO
OH
4a
DMT-Cl
Py
DMT-Cl
Py
O
O
N
N
NH
geranyl bromide
MeOH
DMTO
N
S
DMTO
S
O
O
HO
OH
HO
OH
5
6
NaSeH
EtOH
O
N
O
NH
NH
CH3COOHaq
HO
Se
DMTO
N
Se
O
O
HO
OH
HO
OH
3a
7
Scheme 1. Transformation of S2U 2a to Se2U 3a via an intermediate geS2U 6.
The sodium hydroselenide (NaHSe) required for the subsequent reaction was prepared according to
the published procedure [8] by reduction of elemental selenium with NaBH4 in absolute ethanol
under an inert atmosphere. Importantly, the selenium used in the experiment contained 22 at.% of
the NMR-active 77Se isotope, in contrast to the natural abundance of 77Se isotope, which is
approximately 7.6%. A solution of 77Se-enriched hydroselenide was added to 6, and the mixture was
stirred for 6 h under an inert atmosphere. After aqueous work up, silica gel flash chromatography
furnished 7 as a yellow solid in 30% yield.
The structure of 7 was confirmed by 1H and 13C NMR and by FAB mass spectrometry (Fig. S14 - S17).
The 22% abundance of 77Se retained in 7 was documented by an extended FAB MS spectrum (Fig. 2,
m/z 606.5). The resonance line of 77Se in the labeled uridine (Fig. 3A, 77Se NMR, δ407.4 ppm) was
located downfield of the signal for 77Se-selenourea (δ195 ppm) [9]. 77Se enrichment permitted the
4

acquisition of a 2D HMBC spectrum (Fig. 3B), in which the signal for H1’ at δ6.5 ppm was correlated
with the 77Se signal at δ407.4 ppm.
Compound 7 was then detritylated with aqueous acetic acid (50%) for 1 h at room temperature. The
1
resulting product 3a (Se2U) was isolated by RP-HPLC and characterized by UV (λ 301 nm), H and
max
13C NMR (in D2O), and FAB MS (Fig. S18 – S23). The 77Se isotope contents in 3a and 6 were identical
(Fig. 2 and S24). The 77Se NMR resonance signal for 3a was observed at δ353.7 ppm (Fig. S25), i.e., it
was shifted approximately 54 ppm upfield from the 77Se resonance of 7.
Figure 2. FAB MS spectrum for 7. Signals at m/z 609.3 and m/z 606.5 correspond to the molecules
containing the most abundant 80Se isotope and to those artificially enriched in 77Se isotope,
respectively.
5

A
B
H1`
407.4 ppm
77Se
Figure 3. The 77Se NMR spectrum of 5’-O-DMT-2-selenouridine 7 (A); The two-dimensional HMBC
spectrum showing a cross-peak between H1’ at δ6.5 ppm and 77Se signals at δ(B); 77Se NMR (δ407.4
ppm) for spectrum taken with Ph-Se-Se-Ph reference (δ463 ppm) in CDCl3.
3. Discussion
In this study, we demonstrated that the chemical transformation of S2U to Se2U proceeds easily via
the S-geranylated S2U intermediate, in which substitution at the C2 position with nucleophilic
selenide occurs, accompanied by the departure of the thioalkyl moiety. Under cellular conditions,
this reaction is strictly dependent on the concentration of the selenium donor. At low selenium
concentrations (< 10 nM), the geranylated tRNA form is predominant over the seleno-tRNA form;
however, selenoation occurs at the expense of the formation of geranylated tRNA at selenium
concentrations exceeding 10 nM [4]. Thus, this transformation seems to occur in the cell in a linear
manner via S2U→geS2U→Se2U (Scheme 2) rather than in a parallel manner in which the two
individual reactions S2U→Se2U and S2U→geS2U occur independently, possibly in different catalytic
centers [4, 5].
6

O
N
O
N
O
N
O
N
O
N
3-
NH
N
N
N
NH
Se
Geranyl-pp
SelU
SH
SePO3
SelU
S Geranyl
SeH
S
tRNA
tRNA
tRNA
tRNA
tRNA
Scheme 2. Scheme of a possible cellular pathway of S2U-tRNA to Se2U-tRNA transformation
undergoing through the geS2U-tRNA intermediate (Geranyl-pp – geranyl diphosphate, Se-p –
selenophosphate).
The source of selenium in the S2U to Se2U enzymatic transformation is a selenophosphate (SePO33-),
which is the product of the conversion of selenide (HSe-) by selenophosphate synthetase (SelD)
[10,11]. The reaction utilizes ATP and yields SePO33-, orthophosphate and AMP. Selenophosphate is
also an essential substrate for the generation of selenocysteine (Sec), which is now considered a 21st
amino acid. However, selenocysteine is not formed by the direct exchange of sulfur with selenium in
cysteine but from Ser-tRNASec charged with serine [12,13]. The biosynthesis of Sec begins with
phosphorylation of the serine moiety catalyzed by phosphoseryl-tRNA kinase (PSTK) and yields O-
phosphoseryl-tRNASec [14]. Activated in this way, the hydroxyl group of serine is replaced by selenium
from selenophosphate to afford Sec-tRNASec. This process supports our hypothesis because attack by
the selenium donor is accompanied by the departure of a negatively charged phosphate. From the
other side, the naturally occurring rare nucleoside S-methyl-2-thioadenosine (2-
methylthioadenosine, mS2A) is not the product of consecutive sulfuration and methylation of
adenosine moiety but of a direct transfer of the methylthio group from SAM [15]. In contrast to the
discussed modification there is no any adenosine derivative which would be formed by a nucleophilic
attack at C2 of adenine ring of mS2A with subsequent departure of thiomethyl leaving group. Thus,
more advanced biological studies are required to evaluate the hypothesis that the transformation of
2-thiouridine to 2-selenouridine follows the linear process S2U→geS2U→Se2U.
4. Conclusions
The successful chemical synthesis of Se2U from S2U via S-geranyl-2-thiouridine presented in this
study suggests a possible mechanism of SelU enzyme action in which geS2U serves as an
intermediate product of the cellular transformation of S2U to Se2U.
7

5. Experimental
5. 1. Synthesis of S-geranyl-2-thiouridine (geS2U, 4a) [4]
2-Thiouridine (260.0 mg, 1 mmol) was dissolved in methanol (10 ml). To the solution geranyl bromide
(570 μl, 3 mmol) and N,N-diisopropylethylamine (522 μl, 3 mmol) were added. After stirring at 25°C
for 30 minutes the reaction mixture was concentrated in vacuo. The residue was dissolved in ethyl
acetate and washed with water. The organic layer was dried over anhydrous MgSO4, concentrated in
vacuo and subjected to silica gel chromatography (0-5% methanol in ethyl acetate) to yield a white
1
solid (355.0 mg, 0.89 mmol, 89% yield): TLC Rf = 0.10 (1/9 MeOH/EtOAc (v/v)); H NMR (700 MHz,
CDCl3) δ 8.21 (d, J = 7.6 Hz, 1H), 5.96 (d, J = 7.6 Hz, 1H), 5.91 (d, J = 5.7 Hz, 1H), 5.28 (t, J = 8.1Hz, 1H),
5.06 (t, J = 7.0 Hz, 1H), 4.45 (q, J = 5.2 Hz, 1H), 4.36 (dd, J = 5.2, 2.5 Hz, 1H), 4.19 (q, J = 2.5 Hz, 1H),
3.93 (m, 2H), 3.87 – 3.81 (m, 2H), 2.10 (m, 2H), 2.02 (m, 2H), 1.73 (s, 3H), 1.69 (s, 3H), 1.60 (s, 3H). 13C
NMR (176 MHz, DMSO-d6) δ 166.6, 161.9, 141.1, 139.4, 131.0, 123.7, 117.4, 108.9, 91.3, 85.9, 74.6,
69.9, 60.7, 29.5, 25.9, 25.4, 17.6, 16.1. MS-FAB (m/z): 397.3 [M+H]+, MW: 396.5.
5.2. Synthesis of 5`-O-dimetoxytrityl-S-geranyl-2-thiouridine (6) from 4a
A solution of S-geranyl-2-thiouridine 4a (355.0 mg, 0.89 mmol) and 4,4 -dimethoxytrityl chloride
(603.0 mg, 1.78 mmol) in pyridine (4 ml) was stirred at 25 °C for 20 hours. To the resulting reaction
mixture, 10 ml water was added and extracted with CH2Cl2. Organic layer was dried over anhydrous
MgSO4, concentrated in vacuo and subjected to silica gel chromatography (0-2% methanol in
methylene chloride with 0,5 % Py). A white solid (470.0 mg, 0.67 mmol, 75% yield) of 6 was obtained
after column chromatography: TLC Rf = 0.67 (1/9 MeOH/CHCl3 (v/v)); 1H NMR (700 MHz, CDCl3) δ
7.84 (d, J = 7.7 Hz, 1H), 7.43 (dd, J = 8.3, 1.4 Hz, 2H), 7.38 – 7.16 (m, 13H), 6.91 – 6.86 (m, 4H), 5.92 (d,
J = 6.3 Hz, 1H), 5.57 (d, J = 7.7 Hz, 1H), 5.32 (m, 1H), 5.09 (m, 1H), 4.66 (q, J = 5.6 Hz, 1H), 4.43 (m,
1H), 4.26 (q, J = 2.6 Hz, 1H), 3.98 (dd, J = 12.8, 8.0 Hz, 1H), 3.87 (dd, J = 12.8, 8.0 Hz, 1H), 3.79 (s, 6H),
3.45 (t, J = 2.8 Hz, 2H), 2.18 – 2.08 (m, 2H), 2.09 – 1.98 (m, 2H), 1.76 (s, 3H), 1.71 (s, 3H), 1.63 (s, 3H);
13C NMR (176 MHz, CDCl3) δ 171.20, 166.70, 161.08, 146.69, 145,40, 142.29, 137.46, 134.26, 132.54,
131.41, 130.61, 129.45, 127.68, 126.07, 118.44, 115.75, 111.14, 93.98, 89.53, 87.10, 77.10, 74.25,
65.91, 57.58, 42.05, 34.15, 28.79, 28.08, 20.11, 18.91. MS-FAB (m/z): 699.50 [M+H]+, MW: 698.88.
5.3. Synthesis of 5’-O-dimetoxytrityl-2-selenouridine (7) [8]
Nucleoside 6 (80 mg), NaBH4 and Se were dried overnight under reduced pressure. A solution of
NaSeH was generated by addition of absolute ethanol (5 ml) to selenium (110 mg of selenium of
natural abundance and 23 mg of 77Se) and sodium borohydride (NaBH4, 94 mg) at 0°C. The reaction
8

was completed in 1h and clear solution was formed. The ethanolic solution was added to 6 (80 mg)
and the mixture was stirred for 3h under argon. The reaction mixture was concentrated under
reduced pressure and chloroform (5 ml) was added to the residue. The organic layer was washed
twice with water (2x3 ml), and then dried over anhydrous magnesium sulfate. Purification was
performed by flash chromatography (4% methanol in methylene chloride) and the light yellow
1
compound 7 was obtained (30% yield). TLC Rf = 0.60 (1/9 MeOH/CHCl3 (v/v)); H NMR (500 MHz,
CDCl3) δ 8.61 (s, 1H), 8.26 (d, J = 8.1 Hz, 1H), 7.67 (t, J = 7.6 Hz, 1H), 7.38 – 7.31 (m, 2H), 7.31 – 7.16
(m, 10H), 6.85 – 6.76 (m, 4H), 6.55 (d, J = 1.5 Hz, 1H), 5.58 (d, J = 8.1 Hz, 1H), 4.49 (dd, J = 7.7, 5.3 Hz,
1H), 4.44 (dd, J = 5.3, 1.5 Hz, 1H), 4.19 (dt, J = 7.7, 2.3 Hz, 1H), 3.76 (d, J = 1.9 Hz, 13H), 3.56 (qd, J =
11.3, 2.3 Hz, 2H), 1.29 – 1.16 (m, 5H). 13C NMR (126 MHz, Chloroform-d) δ 175.64, 159.40, 158.72,
149.50, 144.29, 140.73, 136.43, 135.22, 134.97, 130.14, 128.10, 127.24, 123.94, 113.35, 108.14,
96.64, 87.13, 84.05, 68.81, 61.03, 55.29. 77Se NMR (95 MHz, CDCl3) δ 407.45. MS-FAB (m/z): 609.30
[M+H]+, MW: 608.53.
5.4. Synthesis of 2-selenouridine (3a)
2-Selenouridine (3a) was prepared through detritylation of 5`-O-dimethoxytrityl-2-selenouridine (5
mg, 0.008 mmol) by acid treatment (1h, room temperature) (quantitative removal of DMT group).
Then the reaction mixture was concentrated under reduced pressure (SpeedVac) and the remaining
crude 2-selenouridine was purified by RP-HPLC. The product elution was performed with a gradient
of buffer A (20 mM triethylammonium acetate TEAAc, pH 7.1 in water) to buffer B (20mM
triethylammonium acetate TEAAc, pH=7.1 in 50 % acetonitrile) on a Kinetex C18 column (5 μm, 4.6
mm x 250 mm, Phenomenex) at a constant flow rate of 1.0 ml/min (0→15 min from 0 to 25% of B;
1
15→20 min from 25 to 100 % of B). Yield 1.95 mg (ca. 80%). H NMR (600 MHz, D2O) δ 8.03 (dd, J =
8.1, 1.3 Hz, 1H), 6.61 (d, J = 2.5 Hz, 1H), 6.14 (d, J = 8.1 Hz, 1H), 4.29 (q, J = 2.5 Hz, 1H), 4.07 (m, 2H),
3.88 (dd, J = 13.1, 1.6 Hz, 1H), 3.73 (dd, J = 13.1, 2.5 Hz, 1H). 13C NMR (151 MHz, D2O) δ 175.58,
162.83, 141.52, 108.04, 95.86, 83.97, 74.92, 68.33, 59.74. 77Se NMR (95 MHz, D2O) δ 353.79. MS-FAB
(m/z): 307.2 [M+H]+, MW: 306.16.
Acknowledgements
This research was financially supported by The National Science Centre in Poland, Project Number
UMO-2011/03/B/ST5/02669 to B.N. and E.S. for years 2012–2015 and statutory funds of CMMS PAS
and TUL. The authors thank Dr Piotr Guga for the article proofreading.
9

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Figure 2
12

Graphical abstract
Transformation of a wobble 2-thiouridine to 2-selenouridine via S-geranyl-2-thiouridine as a possible
cellular pathway, Paulina Bartos, Anna Maciaszek, Anna Rosinska, Elzbieta Sochacka, and Barbara
Nawrot
Hypothesis: Possible consecutive S2U→geS2U→Se2U
transformation in the cell
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Highlights
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2-Thiouridine is chemically transformed to 2-selenouridine via S-geranyl 2-thiouridine
This pathway is proposed to be possible in the cell
It is suggested that SelU may produce geS2U as an intermediate from S2U to Se2U
2-Selenouridine is characterized by 77Se NMR
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