The influence of the C5 substituent on the 2-thiouridine desulfuration pathway and the conformational analysis of the resulting 4-pyrimidinone products

DOI: 10.1016/j.bmc.2015.07.030

Source and publish data:

Bioorganic and Medicinal Chemistry p. 5587 - 5594 (2015)

Update date:2022-09-26

Topics:

Authors:

Bartos, Paulina

Ebenryter-Olbinska, Katarzyna

Sochacka, Elzbieta

Nawrot, Barbara

Read Full Text PDF DownLoad Join now for total 90,000,000 free articles

Article abstract of DOI:10.1016/j.bmc.2015.07.030

In recent years, increasing attention has been focused on the posttranscriptional modifications present in transfer RNAs (tRNAs), which have been suggested to constitute another level of regulation of gene expression. The most representative among them are the 5-substituted 2-thiouridines (R5S2U), which are located in the wobble position of the anticodon and play a fundamental role in the tuning of the translation process. On the other hand, sulfur-containing biomolecules are the primary site for the attack of reactive oxygen species (ROS). We have previously demonstrated that under in vitro conditions that mimic oxidative stress in the cell, the S2U alone or bound to an RNA chain undergoes desulfuration to yield uridine and 4-pyrimidinone nucleoside (H2U) products. The reaction is pH- and concentration-dependent. In this study, for the first time, we demonstrate that the substituent at the C5 position of the 2-thiouracil ring of R5S2Us influences the desulfuration pathway, and thus the products ratio. As the substituent R changes, the amount of R5H2U increases in the order H- > CH₃O- > CH₃OC(O)CH₂- > HOC(O)CH₂NHCH₂- ≈ CH₃NHCH₂-, and this effect is more pronounced at lower pH. The conformational analysis of the resulting R5H2U products indicates that independent of the nature of the R5 substituent, the R5H2U nucleosides predominantly adopt a C2′-endo sugar ring conformation, as opposed to the preferred C3′-endo conformation of the parent R5S2Us.

Full text of DOI:10.1016/j.bmc.2015.07.030

Bioorganic & Medicinal Chemistry 23 (2015) 5587–5594

Contents lists available at ScienceDirect

Bioorganic & Medicinal Chemistry

journal homepage: w ww.elsevier.com/locate/bmc

The inﬂuence of the C5 substituent on the 2-thiouridine

desulfuration pathway and the conformational analysis of the

resulting 4-pyrimidinone products

Paulina Bartos ^a,, Katarzyna Ebenryter-Olbinska ^a,, Elzbieta Sochacka ^a,, Barbara Nawrot

⇑

^aInstitute of Organic Chemistry, Faculty of Chemistry, Lodz University of Technology, Zeromskiego 116, Lodz, Poland

^bDepartment of Bioorganic Chemistry, Centre of Molecular and Macromolecular Studies, Polish Academy of Sciences, Sienkiewicza 112, 90-363 Lodz, Poland

a r t i c l e i n f o

a b s t r a c t

Article history:

In recent years, increasing attention has been focused on the posttranscriptional modiﬁcations present in

transfer RNAs (tRNAs), which have been suggested to constitute another level of regulation of gene

expression. The most representative among them are the 5-substituted 2-thiouridines (R5S2U), which

are located in the wobble position of the anticodon and play a fundamental role in the tuning of the trans-

lation process. On the other hand, sulfur-containing biomolecules are the primary site for the attack of

reactive oxygen species (ROS). We have previously demonstrated that under in vitro conditions that

mimic oxidative stress in the cell, the S2U alone or bound to an RNA chain undergoes desulfuration to

yield uridine and 4-pyrimidinone nucleoside (H2U) products. The reaction is pH- and concentration-

dependent. In this study, for the ﬁrst time, we demonstrate that the substituent at the C5 position of

the 2-thiouracil ring of R5S2Us inﬂuences the desulfuration pathway, and thus the products ratio. As

the substituent R changes, the amount of R5H2U increases in the order H– > CH₃O– > CH₃OC(O)CH₂– >

HOC(O)CH₂NHCH₂– ꢀ CH₃NHCH₂–, and this effect is more pronounced at lower pH. The conformational

analysis of the resulting R5H2U products indicates that independent of the nature of the R5 substituent,

the R5H2U nucleosides predominantly adopt a C2⁰-endo sugar ring conformation, as opposed to the

preferred C3⁰-endo conformation of the parent R5S2Us.

Received 9 June 2015

Revised 14 July 2015

Accepted 15 July 2015

Available online 22 July 2015

Keywords:

2-Thiouridine

tRNA

Desulfuration

4-Pyrimidinone riboside

Conformational analysis

Oxidative stress

1. Introduction

position of the codon is limited due to the less preferred formation

of a hydrogen bond between the sulfur acceptor of S2U and the

5-Substituted 2-thiouridines (R5S2U) are modiﬁed nucleosides

located in the wobble position of the anticodon (position 34) in

transfer RNAs (tRNAs).¹Their structural features play a fundamen-

tal role in the codon reading and efﬁciency of the translation pro-

cess. These features are related to the presence of a sulfur atom at

position 2 as well as the presence of a substituent at the C5 atom of

the uracil moiety. Replacement of the oxygen atom at C2 with a

sulfur atom shifts the puckering equilibrium of the ribose ring

towards the C3⁰-endo form, which is present in A-type RNA helices

and enhances the base stacking in the RNA chain.²In this confor-

mation, 2-thiouridines predominantly recognize adenosine as the

third letter of the codon via a classical Watson–Crick base pairing,

while the wobble base pairing with a G residue at the third

N1H donor of G.³The inﬂuence of wobble R5S2U nucleosides on

codon–anticodon recognition is strictly dependent on the charac-

teristics of the substituent at the C5 position of the heterobase.^4,5

The role of 5-substituents in tRNA wobble 2-thiouridines/uridines

has been widely studied and has recently been excellently summa-

rized by Takai and Yokoyama,^4awho noted that the electronic

characteristics of the 5-substituent play a role in 5⁰-NNA/G-3⁰

codon reading. All substituents present in wobble uridines/

2-thiouridines which contain X-CH₂– type groups^4aexhibit low

electron-donating characteristics; thus, their contribution to the

electron density of the pyrimidine ring is limited. However,

the situation becomes more complicated when the X residue

contains an amino group, such as in

a methylaminomethyl

(CH₃NHCH₂–, mnm) substituent. According to the suggestion of

Takai and Yokoyama, such a substituent can be protonated under

physiological pH. Therefore, the preferred formation of the

tautomeric structures of 5-substituted uridines/2-thiouridines is

observed in which the positively charged protonated

5-CH₃NH(H⁺)CH₂group is accompanied by the negatively

⇑

Corresponding authors. Tel.: +48 42 680 32 48; fax: +48 42 681 54 83 (B.N.);

tel.: +48 42 631 31 55 (E.S.).

E-mail addresses: elzbieta.sochacka@p.lodz.pl (E. Sochacka), bnawrot@cbmm.

lodz.pl (B. Nawrot).

Equal contribution.

http://dx.doi.org/10.1016/j.bmc.2015.07.030

5588

P. Bartos et al. / Bioorg. Med. Chem. 23 (2015) 5587–5594

charged sulfur in the thiolate residue of the heterobase (Fig. 1). In

this case, the heterobase moiety adopts a double bond system,

which provides two acceptors, one at the O(4) atom and one at

the N(3) atom, that are available for the formation of the two

hydrogen bonds with a complementary unit. Interestingly, the

same nucleobase double bond pattern is present in the recently

discovered S-geranyl 2-thiouridines (R5geS2Us, where R = mnm

or carboxymethylaminomethyl (HOC(O)CH₂NHCH₂–, cmnm))⁶in

which the alkylated sulfur atom of 2-thiouridine leads to the

replacement of the N(3)H donor with the N(3) acceptor center

(Fig. 1). These two types of wobble nucleosides, R5S2U and

R5geS2U, are present in the ﬁrst letter of the anticodon of the

tRNA speciﬁc for glutamine, glutamic acid and leucine and they

preferentially recognize the 3⁰-G-ending codons. In contrast, 3⁰-A-

ending codons are preferentially read by R5S2U wobble units with

R = H, methylcarboxymethyl (CH₃OC(O)CH₂–, mcm) or car-

boxymethyl (HOC(O)CH₂–, cm) substituents. We have recently

published data on the preferred pathway of the desulfuration reac-

tion performed on 2-thiouridine, either alone or assembled into the

RNA chain, demonstrating that the 4-pyrimidinone riboside (H2U,

3a, R = H, Fig. 1) is the main product of this transformation.^7,8This

nucleoside contains a double bond system similar to the zwitteri-

onic form of R5S2U as well as R5geS2U units, where R = (c)mnm,

and forms more stable RNA duplexes when base-paired with G,

and not with A.⁹

H⁺

CH₂

S^-

H⁺

Z=CH₃

1d, R=CH₂NHCH₂COOH (cmnm)

1e, R=CH₂NHCH₃(mnm)

Z=CH₂COOH

R5geS2U

3a, R=H

R=CH₂NHCH₂COOH (cmnm)

R=CH₂NHCH₃(mnm)

Figure 1. Structure of modiﬁed nucleosides S2U, geS2U and H2U.

An efﬁcient transformation of S2U to H2U occurs under in vitro

conditions that mimic oxidative stress in the cell (100 mM H₂O₂)

and is pH- and concentration-dependent.^8,10The preliminary data

indicate that the RNA chain containing 4-pyrimidinone riboside is

prone to depyrimidination and sugar–phosphate backbone cleav-

age.^7,11We hypothesize that the sulfur atom in S2U-tRNAs may

be a primary site for ROS attack in the cells. The resulting major

H2U-tRNA lesion, in addition to changing the A to G codon reading

preference, might be a site of RNA chain cleavage via abasic RNA

intermediate.^12,13This hypothesis, although already conﬁrmed by

in vitro experiments, has not been proven in the cell, and studies

focused on the effect of S2U-tRNA damage on cellular oxidative

stress are currently being performed in our laboratory. One cannot

exclude the fact that the ‘chemically driven’ tRNA damage is

accompanied by anticodon-speciﬁc nuclease-assisted cleavage

induced by oxidative stress, as has been demonstrated in the

literature.^9,14

[O]

2a-e

3a-e

1a-e

R = a) -H b) -OCH₃(mo) c) -CH₂COOCH₃(mcm)

d) -CH₂NHCH₂COOH (cmnm) e) -CH₂NHCH₃(mnm)

Scheme 1. General scheme of desulfuration of 2-thiouridines 1a–e with hydrogen

peroxide in aqueous solution.

In this study, we discuss the inﬂuence of the C5 substituent on

the R5S2U desulfuration pathway performed in the presence of

aqueous H₂O₂, which are conditions that mimic oxidative stress

in the cell (Scheme 1). For the studies, the 5-substituents differ-

ently affecting the decoding properties of the wobble 2-thiouridi-

nes were selected, as mcm and H, which preferentially recognize

the 3⁰-A-ending codons, and mnm and cmnm which recognize also

the 3⁰-G-ending codons. In addition, a 2-thioanalog of the wobble

5-methoxyuridine (mo5U) was also selected (non-identiﬁed in

the natural tRNA) to study the inﬂuence of the X-O- type sub-

stituent on the evaluated process. The ratio of the R5U/R5H2U

reaction products was determined by ¹H NMR measurements

and was found to be strongly dependent on the characteristics of

the C5 substituent and the reaction conditions (water or buffered

aqueous solutions of pH 6.6–7.6). The availability of detailed spec-

tral and conformational data (evaluated based on the values of the

¹H–¹H and ¹H–¹³C coupling constants) of a set of 4-pyrimidinone

nucleosides 3a–e obtained independently (under ‘organic condi-

tions’), as well as the respective spectral and conformational data

for the parent 5-substituted 2-thiouridines 1a–e and uridines

2a–e (obtained in house) enabled a complete comparable confor-

mational analysis. The results aided in the elucidation of how the

loss of the 2-thiocarbonyl/carbonyl function changes the structural

and electronic features of the resulting 4-pyrimidinone nucleosides

in solution and how these features may inﬂuence the hybridization

properties of RNA duplexes containing these units.

2. Results and discussion

2.1. Chemistry

In our previous studies, we have shown that the 5⁰,3⁰,2⁰-O-ben-

zoylated derivative of 2-thiouridine could be exclusively trans-

formed into H2U (3a, R = H) under ‘organic conditions’ by

treating the starting 2-thiouridine derivative with trans-2-

phenylsulfonyl-3-phenyloxaziridine (PSO) in pyridine or with

meta-chloroperbenzoic acid (mCPBA) in a mixture of dichloro-

methane/pyridine at room temperature.¹⁵m-Chloroperbenzoic

acid was also successfully applied to the selective oxidative desul-

furation of 5⁰-DMT (4,4⁰-dimethoxytrityl)-protected 2⁰-deoxy-5-

methyl-2-thiouridine.¹⁶

In this study, we successfully applied this ‘organic’ methodol-

ogy to the oxidative desulfuration of the per-O-benzoylated

derivatives of 5-methoxy-2-thiouridine (mo5S2U) 4b and

5-methylcarboxymethyl-2-thiouridine (mcm5S2U) 4c (Scheme 2A)

as well as to the 2⁰,3⁰-O-isopropylidene derivative of N-COCF₃-pro-

tected cmnm5S2U 6d and the 2⁰,3⁰-O-isopropylidene derivative of

P. Bartos et al. / Bioorg. Med. Chem. 23 (2015) 5587–5594

5589

These syntheses were repeated, the sets of 1a–e and 2a–e were

obtained, and their structures were conﬁrmed. The respective ¹H

NMR spectra of compounds used in these studies are given in

Supplementary material.

(A)

BzO

2.2. Conformational analysis of 5-substituted 4-pyrimidinone

nucleosides

MeoNa

MeOH

mCPBA

OBz OBz

Generally, nucleoside conformation is described relative to three

different aspects, namely (i) the dominant sugar ring pucker, (ii) the

preferred glycosidic bond arrangement and (iii) the orientation of

the 5⁰-hydroxyl group against the furanose ring.¹⁹In the present

studies, a conformational analysis of 5-substituted 4-pyrimidinone

nucleosides 3, as well as their parent 2-thiouridines 1 and uridines

2 was performed based on the NMR data collected in the aqueous

solutions.

3a, 3b, 3c

4a, 4b, 4c

5a, 5b, 5c

R= a) -H b) -OCH₃c) -CH₂COOCH₃

(B)

R₁

NH₃

MeOH

2.2.1. Conformation of the sugar moiety

1) CH₃COOH

It is assumed that the sugar ring of the nucleoside in solution

exists as an equilibrium mixture of the two puckered forms:

C2⁰-endo (S conformer) or C3⁰-endo (N conformer).²⁰The percent-

age of S and N conformers can be estimated based on the values

2) mCPBA

of the ³J_{H1 –H2}and ³J_{H3 –H4}coupling constants according to the fol-

3d, 3e

R= CH₂NHCH₂COOH (3d)

6d, 6e

7d, 7e

lowing equations:% C2⁰-endo = 100 J_{H1 –H2}/(J_{H1 –H2}+ J_{H3 –H4}) and %

R₁= d) -CH₂N(COCF₃)CH₂COOH

e) -CH₂N(COCF₃)CH₃

C3⁰-endo = 100 – % C2⁰-endo.²¹Thus, ¹H NMR data were collected,

CH₂NHCH₃

(3e)

and experimental J_H–Hcoupling constants were extracted for all

(C)

of the screened compounds. These data were used for the calcula-

tion of a population of C3⁰-endo conformers of 4-pyrimidinone

nucleosides 3 and their parent 2-thiouridines 1 and uridines 2

for the R substituents abbreviated as a–e as well as c⁰and c⁰⁰

(Table 1).

H₂N C CH₂

H₃CO C CH₂

HO C CH₂

Previous studies have shown that the population of the C3⁰-endo

conformer of 2-thiouridines is markedly higher than that of the

analogous uridines.^2b,c,22The rigidity of the C3⁰-endo form of S2U

is caused by the steric effect of the large 2-thiocarbonyl group of

the heterobase and the 2⁰-hydroxyl group of the ribose ring.²³

This intrinsic conformational rigidity strongly contributes to the

stability of the duplexes containing S2U-modiﬁed strands, thereby

inﬂuencing the decoding process performed by 2-thiouridine-

modiﬁed tRNA.²⁴Deprivation of the sulfur moiety in 2-thiouridine

1 leads to a dramatic change in the sugar ring puckering in the

resulting H2U nucleosides 3. This is observed for all seven cases

analyzed (Table 1) for which 2-thiouridines 1a–e adopt the

C3⁰-endo conformation in 71–82%, while their desulfuration prod-

ucts 3a–e adopt the C3⁰-endo conformation only in 37–43%. This

result indicates that the C2⁰-endo conformation is dominant in the

equilibrium of the sugar conformers of 4-pyrimidinone nucleosides

in solution. Interestingly, the substituent at the C5 position alters

the sugar ring puckering to a lesser extent, although it is clearly

observed that in the case of mo5H2U, the more electron-donating

characteristics of the methoxyl substituent shifts the sugar ring

puckering equilibrium towards the C3⁰-endo conformation (43% of

3b compared with 37–39% of the remaining R5H2Us studied).

NH₃

0.1 M KOH

3c'

3c"

Scheme 2. Synthetic pathways for the preparation of 5-substituted 4-pyrimidinone

nucleosides 3b–e, starting either from perbenzoylated derivatives (A) or N-

protected 2⁰,3⁰-O-isopropylidene derivatives of 5-substituted-2-thiouridines b–e

(B) (the descriptions are in Scheme 1). The synthesis of amido- and carboxy-

derivatives of 3c (3c⁰and 3c⁰⁰, respectively) is shown in Scheme C.

N-COCF₃-protected mnm5S2U 6e (Scheme 2B). Further deprotec-

tion procedures under alkaline conditions afforded the ﬁnal 5-sub-

stituted 4-pyrimidinone nucleosides 3b–e, where R = mo (b), mcm

(c), cmnm (d) or mnm (e). Additionally, 4-pyrimidinone nucleoside

3c with a methyl ester functionality in the 5-substituent was trans-

formed into the corresponding amide and carboxylic derivatives

3c⁰and 3c⁰⁰, respectively (Scheme 2C). All of the 4-pyrimidinone

nucleosides were puriﬁed by silica gel column chromatography

and were obtained as white foams that were characterized by

spectral analysis (¹H and ¹³C NMR and fast atom bombardment

mass spectrometry (FAB-MS)). The obtained data are included

either in Experimental section or in Supplementary material.

Note that that 3a–c, 3c⁰and 3c⁰⁰were sufﬁciently stable in aqueous

solution, while 3d and 3e underwent decomposition under the

same conditions. Due to the instability of 3d and 3e, only limited

spectral analysis of these nucleosides could be performed.

2.2.2. Conformation around the glycosidic bond

The syn/anti conformation around the N-glycosidic bond was

investigated using vicinal carbon–proton coupling. Proton-coupled

¹³C spectra reveal carbon–proton couplings that indicate the

syn/anti conformation according to the following relationships:

The required substrates, 2-thiouridines 1a–e, as well as their

parent uridine derivatives 2a–e, are known compounds, and their

syntheses were previously established either in our laboratory or

elsewhere using either the methodology of N-glycosidic bond for-

mation (nucleosides with R = H, mo, mcm)^2a,17or by the

introduction of a 5-substituent into the appropriate derivative of

2-thiouridine/uridine (nucleosides with R = mnm and cmnm).¹⁸

J_C6–H1> J_C2–H1for the anti conformation and J_C2–H1> J_C6–H1for

the syn conformation. The data presented in Table 2 indicate that

the 3a–c H2U units adopt a slightly prevailing syn conformation

around the N-glycosidic bond, in contrast to the predominant anti

conformation of the parent R5S2Us and R5Us.²⁵This observed fea-

ture originates from the lack of the sulfur/oxygen moiety at C2 of

the 2-thiouracil/uracil ring, which ensures fast rotation around

5590

P. Bartos et al. / Bioorg. Med. Chem. 23 (2015) 5587–5594

Table 1

Table 3

Ribose ring puckering calculated from the coupling constants (J in Hz)

Population of the conformers of R5H2U nucleosides around the C4⁰–C5⁰bond

Nucleoside

Number

C3⁰-endo (%)

Nucleoside

% g⁺

% t

% g^ꢁ

J_{H1 –H2}

J_{H3 –H4}

J_{H4 –H5}

S2U

H2U

2.5

4.8

5.7

6.0

5.4

3.5

H2U (3a)

3.1

2.9

3.1

3.0

4.0

3.3

4.0

4.1

3.9

mo5H2U (3b)

mcm5H2U (3c)

ncm5H2U (3c⁰)

cm5H2U (3c⁰⁰)

mo5S2U

mo5U

mo5H2U

1.8

4.1

5.3

8.1

5.6

4.0

mcm5S2U

mcm5U

mcm5H2U

2.3

4.5

5.8

7.8

5.7

3.6

similar to the prevailing structure of R5S2Us and R5Us in

aqueous solutions.

ncm5S2U

ncm5U

ncm5H2U

1c⁰

2c⁰

3c⁰

2.0

4.4

5.8

7.4

5.5

3.7

The obtained data for the sugar ring conformation of H2U in

solution are in agreement with those obtained for the H2U crystal

structure.²⁹In the crystal, the H2U nucleoside exclusively adopts

the C2⁰-endo sugar ring conformation. The above conformational

analysis of the R5H2U units clearly demonstrates that the sub-

stituents at C5 on the 4-pyrimidinone ring have only a slight effect

on their structure. The only substituent that inﬂuences the confor-

mational parameters slightly more than the others is the 5-meth-

oxyl group in mo5H2U for which the highest population of the

C3⁰-endo, syn and g⁺conformer is observed compared with the

other R substituents.

cm5S2U

cm5U

cm5H2U

1c⁰⁰

2c⁰⁰

3c⁰⁰

2.3

4.4

5.8

7.5

5.6

3.7

cmnm5S2U

cmnm5U

cmnm5H2U

1.7

4.2

5.7

7.7

5.7

3.6

mnm5S2U

mnm5U

mnm5H2U

2.4

4.2

5.9

7.7

5.6

3.4

To summarize this conformational analysis, we demonstrate for

the ﬁrst time that independent of the nature of the R5 substituent,

the R5H2U nucleosides predominantly adopt a C2⁰-endo sugar ring

conformation. This feature, along with the restructured hydrogen

bond donor/acceptor pattern, has dramatic consequences on the

codon–anticodon interactions if these H2U-type nucleosides are

located in a wobble position of the anticodon, as in the case for

those mentioned above, that is, (c)mnm5geS2U. NMR data for

the geS2U nucleoside obtained by Dumelin et al.⁶and by our

group³⁰have shown that this nucleoside preferentially adopts a

C2⁰-endo sugar ring conformation. The respective coupling con-

the N-glycosidic bond; thus, similar populations of both of the pos-

sible conformers are feasible. Therefore, there is no signiﬁcant

preference of one, neither syn nor anti, conformation. Again, the

5-methoxyl substituent that has more electron-donating charac-

teristics than hydrogen drives the nucleobase towards the syn con-

formation (34% of the anti conformation of mo5H2U compared

with 48% of H2U).

2.2.3. Conformation around the C4⁰–C5⁰bond

The local conformation around the C4⁰–C5⁰bond was examined

by analyzing the contribution of each of the three distinct forms,

gauche (+), trans, and gauche (ꢁ), using the coupling constants of

the H5⁰and H5⁰⁰protons with H4⁰.^25a,26The absolute assignment

of the H5⁰and H5⁰⁰protons in the ¹H NMR spectra was evaluated

using the rules elaborated based on the deshielding effect of the

phosphate group on H5⁰and H5⁰⁰in the 3⁰-monophosphates of

the uridines.²⁷It was shown that the H5⁰and H5⁰⁰spectral regions

show similar characteristic signal patterns, d(H5⁰) > d(H5⁰⁰) and

stants J_{1 ,2}are 5.4⁶and 5.7 Hz,⁸respectively, in a similar range to

those assigned in the reported studies (J_{1 ,2}= 5.3–5.9 Hz for

R5H2U units, see Table 1). Note that in the Yokoyama model of

wobble recognition,^4athe protonated mnm5S2U nucleoside is

suggested to preferentially read 3⁰-G-ending codons via a H2U-

type structure in the wobble base pair, assuming that the wobble

nucleoside is constrained in the C3⁰-endo conformation. Our

results, however, show that the H2U-type units preferentially

adopt the C2⁰-endo sugar ring puckering, suggesting that in this

conformation, H2U-type thiouridines are more ﬂexible and prefer

the decoding of guanosine at the 3⁰-end of the codon according

to the wobble mode.

J_{H4 –H5}< J_{H4 –H5}

number of other nucleosides and

nucleotides.^25a,28Therefore, it appears reasonable to assume that

for all of these nucleosides, where this pattern is observed, the

more shielded proton is assigned as H5⁰⁰. The speciﬁc

assignments of the H5⁰and H5⁰⁰methylene protons in the ¹H

NMR spectra of 4-pyrimidinone nucleosides 3a–c and the

2.3. Desulfuration of 5-substituted 2-thiouridines in aqueous

buffered solutions

measurements of the J_{H4 –H5}and J_{H4 –H5}proton–proton coupling

constants enabled the determination of the populations of the

three exocyclic C4⁰–C5⁰rotamers gauche (+) (g⁺), trans (t) and

gauche (ꢁ) (g^ꢁ) (Table 3). The obtained data demonstrate that for

the R5H2U nucleosides, the prevailing conformation around the

C4⁰–C5⁰bond is the one described as gauche (+). This feature is

To understand the inﬂuence of the C5 substituent on the desul-

furation pathway of the R5S2U units under conditions that may

occur during oxidative stress in the cell, desulfuration reactions

were performed in the presence of aqueous H₂O₂, either in water

or in phosphate buffer (pH 6.6, 7.2 and 7.6), at 25 °C (Scheme 1).

The progress of the reaction was monitored by ¹H NMR spec-

troscopy. For this reaction, 10 mM solutions of R5S2U substrate 1

and 100 mM H₂O₂were used to achieve sufﬁcient substrate trans-

formation and to ensure the proper ¹H signal integration required

to monitor the ratio of the reaction mixture components. Figure 2

represents an example of a set of three spectra for S2U 1a, U 1b and

H2U 1c and the signals (preferably H6) that were used to evaluate

the ratio of the reaction mixture components (description of the

spectra are provided in Supplementary material, Fig. S1). The

remaining sets of spectra for R5S2U, R5U and R5H2U, where

R = mcm, mnm, cmnm and mo, are given in Figures S2–S5.

Table 2

Population of the conformers of R5H2U nucleosides around the N-glycosidic bond

Nucleoside

Number

J_C2–H1

J_C6–H1

% anti/syn

H2U

3c⁰

3c⁰⁰

3.4

3.8

3.6

3.5

4.0

3.7

3.6

3.8

48:52

34:66

42:58

45:55

43:57

mo5H2U

mcm5H2U

ncm5H2U

cm5H2U

P. Bartos et al. / Bioorg. Med. Chem. 23 (2015) 5587–5594

5591

Figure 2. ¹H NMR spectra of S2U, U and H2U collected in D₂O.

First, we monitored the progress of the desulfuration reaction

under three different pH conditions, at 6.6, 7.2 and 7.6. At given

time intervals, the ¹H NMR spectra were collected (Figs. S6–S20),

and the k_obsvalues were calculated from the pseudo ﬁrst-order

equation k_obs¼ A₀e^ꢁkxþ y₀. The values of k_obsand t_1/2for the desul-

furation reactions of 1a–e in pH 6.6, 7.2 and 7.6 are given in

Table 4. The lowest reaction rate at each monitored pH is observed

for the desulfuration of S2U itself, with R being a hydrogen atom.

This is also conﬁrmed by the longest half-life (t_1/2) for S2U disap-

pearance, 78.41, 23.74 and 9.11 min at pH values of 6.6, 7.2 and

7.6, respectively. Interestingly, the highest desulfuration rates

and the smallest respective half-lives are observed for the reac-

tions performed under the most basic conditions (pH 7.6). At the

lowest pH (6.6), the most efﬁcient reaction was the desulfuration

of mo5S2U for which the t_1/2value was 20.49 min, and this value

decreased ca. 2.5-fold at the highest pH (at pH 7.6, t_1/2was

8.8 min). The rate of this reaction was less dependent on the pH,

while the other desulfuration reactions were more sensitive to

pH, as demonstrated by a shortening of their t_1/2values at pH 7.6

by ca. 6–9 times relative to those at pH 6.6. The reactions required

10–160 min for completion. The results given in Table S1 and pre-

sented in Figure 3 indicate that the desulfuration reaction of R5S2U

is both pH- and C5-substituent dependent.

At a lower pH (6.6), each of the screened substrates delivered a

4-pyrimidinone derivative, and when the C5 substituents were

hydrogen, methoxyl or methylcarboxymethyl groups, the R5H2Us

were the prevailing reaction products (3a–c, R = H, mo and

mcm). In contrast, mnm5H2U (3e) and cmnm5H2U (3d) comprised

only ca. 30% of the reaction products. An increase in pH for the

desulfuration reaction resulted in the lowering of the contribution

of the 4-pyrimidinone nucleosides in the reaction mixture, and this

effect was the most visible for desulfuration of mnm5S2U (1e) and

cmnm5S2U (1d). The remaining substrates were still partially

transformed to products 3 at pH 7.2 and 7.6.

electron densities on the C2 sulfur atom (causing the different

reactivity of C2 sulfur atom). The electron-donating 5-methoxy

substituent (mo) inﬂuences the heterobase (pyrimidine) electron

density via a mesomeric effect that directs the reaction pathway

to the more preferable H2U nucleoside formation under the phys-

iological pH range. Note, that 2-thiouridine with a 5-methoxy sub-

stituent at the pyrimidine 5 position has not been found in nature,

and only 5-methoxyuridine was identiﬁed at the wobble position

of Bacillus subtilis tRNAs speciﬁc for Thr, Ala and Pro.³¹

In the case of the remaining substituents connected to the

pyrimidine ring by the methylene group, the resonance effect is

relatively small, and the inductive effect may play a dominant role,

especially for substituents that can be protonated, thus altering

their electron-withdrawing properties. The partial protonation of

the amine function of the cmnm and mnm substituents of 1d

and 1e at physiological pH causes the localization of a partial neg-

ative charge on the sulfur atom, which promotes faster oxidation

to 5-cmnm- and 5-mnm-uridine products 2d and 2e, respectively.

This transformation should be less dangerous for cell metabolism.

Interestingly, the H2U lesion is observed only for the transforma-

tion that occurs at a lower pH (6.6), which may appear in cancer-

changed cells. One may conclude that oxidative stress in cancer

cells induces further lesions, leading to further alterations of cell

metabolism.

2.3.1. Desulfuration of 5-substituted 2-thiouridines in water

It is important to note that the release of the sulfate oxygen dur-

ing the reaction causes an acidiﬁcation of the reaction mixture to

pH 3–4.⁷Therefore, we monitored the direction of the desulfura-

tion pathway for the reactions performed also in water.

Substrates 1a–e in water solutions were treated with H₂O₂, and

after the reaction completion, the ratio of the products was deter-

mined by ¹H NMR integration. At a low pH, all of the reactions

yielded the prevailing 4-pyrimidinone products (Table 5), except

of desulfuration of mnm5S2U 1e, which resulted in the prevailing

mnm5U product (59%) against mnm5H2U 3e (41%). Also it was vis-

ible, that 1d (cmnm5S2U) gave more cmnm5U (2d) product than

1a,b and c, but less than latter one, although the 4-pyrimidinone

congener 3d was still the major one (69%).

Our results clearly demonstrate that the replacement of the

hydrogen atom at the 5 position of 2-thiouridine by a substituent

with different electronic properties changes the reactivity towards

oxidation agents. This is due to the different electron distributions

within the

p system of the heterobase ring and the different

5592

P. Bartos et al. / Bioorg. Med. Chem. 23 (2015) 5587–5594

Table 4

Values of k_obs(min^ꢁ1ꢂ 10^ꢁ3) and t_1/2(min) for the desulfuration reactions of 1a–e at pH 6.6, 7.2 and 7.6

Substrate

pH 6.6

pH 7.2

pH 7.6

k_obs[min^ꢁ1ꢂ 10^ꢁ3

]

t_1/2(min)

k_obs[min^ꢁ1ꢂ 10^ꢁ3

]

t_1/2(min)

k_obs[min^ꢁ1ꢂ 10^ꢁ3

]

t_1/2(min)

S2U (1a)

8.84 0.38

33.83 5.00

15.95 1.61

21.85 2.98

22.01 2.73

78.41

20.49

43.46

31.72

31.49

29.20 5.00

61.24 7.99

52.24 5.35

55.55 4.86

80.78 6.13

23.74

11.32

13.27

12.48

8.58

76.06 8.17

78.77 2.20

118.37 13.70

132.99 12.50

156.98 6.37

9.11

8.80

5.96

5.21

4.42

mo5S2U (1b)

mcm5S2U (1c)

cmnm5S2U (1d)

mnm5S2U (1e)

H₃C

COCF₃

H₂O₂

1e'

3e'

2e'

Scheme 3. Transformation of N-protected mnm5S2U to its desulfured mnm5U and

mnm5H2U products.

3. Conclusions

Although 5-substituents of 2-thiouridines do not directly

participate in reactions with oxidizers, they strongly inﬂuence

the oxidation process, and the differences in product distribution

(H2U-type nucleosides versus uridines). The amount of R5H2U

increases in the order H > mo > mcm > cmnm ꢀ mnm, and this

effect is observed within the physiological range of one pH unit

between pH 6.6 and 7.6. Protonation of the (c)mnm substituent

in (c)mnm5S2U is a decisive parameter of the transformation path-

way. The conformational analysis of the resulting R5H2U products

indicates that independent of the nature of the R5 substituent, the

R5H2U nucleosides predominantly adopt a C2⁰-endo sugar ring

conformation, as opposed to the preferred C3⁰-endo conformation

of the parent R5S2Us. Our results contribute to the extension of

knowledge on the function of 5-substituted 2-thiouridines in read-

ing the genetic information.

Figure 3. The yields of the R5H2U products for the reactions performed under three

different pH conditions, at 6.6, 7.2 and 7.6. R = H, mo, mcm, cmnm, mnm.

Probably, in such a low pH, the amino function of 1e is almost

fully protonated and this electronic feature of the nucleobase inﬂu-

ences the reaction pathway. To prove this assumption, that is the

contribution of the side chain in the desulfuration pathway of

mnm5S2U we blocked its amine function with a triﬂuoroacetic

group (CF₃CO) (Scheme 3, compound 1e⁰).

4. Experimental

The desulfuration of 1e⁰in water was performed under analo-

gous conditions and delivered the respective 4-pyrimidinone pro-

duct 3e⁰with 88% yield. This result clearly indicates that the

blocking of the amine function of the mnm substituent prevents

its protonation and changes the pathway of the desulfuration reac-

tion. Thus, an earlier suggestion by Yokoyama and Takai^4athat NH

protonation of 1e may contribute to charge distribution in the

mnm5S2U nucleoside is proved here, by the results shown in

this study. This phenomenon most likely contributes to the fact

that only 2-thiouridines substituted with mnm or cmnm are

shown to be geranylated in a natural system, and this conﬁrms

also that the sulfur atom in these two nucleosides is more

electronegative, comparing other 5-substituted 2-thiouridines.

4.1. General procedure for the synthesis of 5-substituted

4-pyrimidinone nucleosides (3a–e)

4.1.1. Procedure (A) for 3b and 3c

Sugar-benzoylated 2-thionucleoside (0.5 mmol) (4b, R = –OCH₃

or 4c, R = –CH₂COOCH₃, according to Scheme 2) was dissolved in

anhydrous CH₂Cl₂(4 ml), and then 0.2 M mCPBA in CH₂Cl₂(4 ml)

was added. The solution was stirred at room temperature, and after

3 h, the reaction was judged to be complete (TLC, 2% MeOH in

CHCl₃). Then, the reaction mixture was washed with 10% Na₂SO₃

(15 ml), washed with 5% NaHCO₃(15 ml), dried over MgSO₄and

evaporated. The ﬁnal solution was ﬁltered off, evaporated in vacuo

and puriﬁed by silica gel column chromatography (2% MeOH in

CHCl₃) to give 5b in 79% yield and 5c in 77% yield.

Table 5

The ratio of R5H2U to R5U products of the desulfuration reaction of R5S2Us

performed in water

Removal of the protecting groups

The benzoylated derivative 5b or 5c (0.4 mmol) was suspended

in 1 M MeONa in MeOH (5 ml). The mixture was stirred at room

temperature for 2 h. Then, TLC analysis (20% MeOH in CHCl₃)

showed that the starting material was completely consumed. The

mixture was evaporated in vacuo, and the oily residue was puriﬁed

by silica gel column chromatography with CHCl₃/MeOH (a gradient

S2U (3a/2a)